Activated Schwann cells put out cytoplasmic processes that play a significant role in cell migration and axon regeneration. Following nerve injury, axonal mitochondria release mitochondrial damage-associated molecular patterns (mtDAMPs), including formylated peptides and mitochondrial DNA (mtDNA). We hypothesize that mtDAMPs released from disintegrated axonal mitochondria may stimulate Schwann cells to put out cytoplasmic processes. We investigated RT4-D6P2T schwannoma cells (RT4) in vitro treated with N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP) or cytosine-phospho-guanine oligodeoxynucleotide (CpG ODN) for 1, 6 and 24 h. We also used immunohistochemical detection to monitor the expression of formylpeptide receptor 2 (FPR2) and toll-like receptor 9 (TLR9), the canonical receptors for formylated peptides and mtDNA, in RT4 cells and Schwann cells distal to nerve injury. RT4 cells treated with fMLP put out a significantly higher number of cytoplasmic processes compared to control cells. Preincubation with PBP10, a selective inhibitor of FPR2 resulted in a significant reduction of cytoplasmic process outgrowth. A significantly higher number of cytoplasmic processes was also found after treatment with CpG ODN compared to control cells. Pretreatment with inhibitory ODN (INH ODN) resulted in a reduced number of cytoplasmic processes after subsequent treatment with CpG ODN only at 6 h, but 1 and 24 h treatment with CpG ODN demonstrated an additive effect of INH ODN on the development of cytoplasmic processes. Immunohistochemistry and western blot detected increased levels of tyrosine-phosphorylated paxillin in RT4 cells associated with cytoplasmic process outgrowth after fMLP or CpG ODN treatment. We found increased immunofluorescence of FPR2 and TLR9 in RT4 cells treated with fMLP or CpG ODN as well as in activated Schwann cells distal to the nerve injury. In addition, activated Schwann cells displayed FPR2 and TLR9 immunostaining close to GAP43-immunopositive regenerated axons and their growth cones after nerve crush. Increased FPR2 and TLR9 immunoreaction was associated with activation of p38 and NFkB, respectively. Surprisingly, the growth cones displayed also FPR2 and TLR9 immunostaining. These results present the first evidence that potential mtDAMPs may play a key role in the induction of Schwann cell processes. This reaction of Schwann cells can be mediated via FPR2 and TLR9 that are canonical receptors for formylated peptides and mtDNA. The possible role for FPR2 and TLR9 in growth cones is also discussed.

Department of Anatomy Division of Neuroanatomy Faculty of Medicine Masaryk University Brno Czechia

Zobrazit více v PubMed

Achuthan A., Elsegood C., Masendycz P., Hamilton J. A., Scholz G. M. (2006). CpG DNA enhances macrophage cell spreading by promoting the Src-family kinase-mediated phosphorylation of paxillin. Cell. Signal. 18, 2252–2261. 10.1016/j.cellsig.2006.05.007 PubMed DOI

Avalos A. M., Ploegh H. L. (2011). Competition by inhibitory oligonucleotides prevents binding of CpG to C-terminal TLR9. Eur. J. Immunol. 41, 2820–2827. 10.1002/eji.201141563 PubMed DOI PMC

Boerboom A., Dion V., Chariot A., Franzen R. (2016). Molecular mechanisms involved in Schwann cell plasticity. Front. Mol. Neurosci. 10:38. 10.3389/fnmol.2017.00038 PubMed DOI PMC

Boivin A., Pineau I., Barrette B., Filali M., Vallières N., Rivest S., et al. . (2007). Toll-like receptor signaling is critical for Wallerian degeneration and functional recovery after peripheral nerve injury. J. Neurosci. 27, 12565–12576. 10.1523/JNEUROSCI.3027-07.2007 PubMed DOI PMC

Cattaneo F., Guerra G., Ammendola R. (2010). Expression and signaling of formyl-peptide receptors in the brain. Neurochem. Res. 35, 2018–2026. 10.1007/s11064-010-0301-5 PubMed DOI

Chen J. J., Nag S., Vidi P. A., Irudayaraj J. (2011). Single molecule in vivo analysis of toll-like receptor 9 and CpG DNA interaction. PLoS One 6:e17991. 10.1371/journal.pone.0017991 PubMed DOI PMC

Coleman M. (2005). Axon degeneration mechanisms: commonality amid diversity. Nat. Rev. Neurosci. 6, 889–898. 10.1038/nrn1788 PubMed DOI

De S., Trigueros M. A., Kalyvas A., David S. (2003). Phospholipase A2 plays an important role in myelin breakdown and phagocytosis during Wallerian degeneration. Mol. Cell. Neurosci. 24, 753–765. 10.1016/s1044-7431(03)00241-0 PubMed DOI

Deumens R., Bozkurt A., Meek M. F., Marcus M. A. E., Joosten E. A. J., Weis J., et al. . (2010). Repairing injured peripheral nerves: bridging the gap. Progr. Neurobiol. 92, 245–276. 10.1016/j.pneurobio.2010.10.002 PubMed DOI

Dubový P. (2017). “Cytokines and their implication in axon degeneration and regeneration following peripheral nerve injury,” in Cytokine Effector Functions in Tissues, eds Foti M., Locati M. (Cambridge, MA: Academic Press; ), 139–148.

Dubový P., Brázda V., Klusáková I., Hradilová-Svíženská I. (2013). Bilateral elevation of interleukin-6 protein and mRNA in both lumbar and cervical dorsal root ganglia following unilateral chronic compression injury of the sciatic nerve. J. Neuroinflammation 10:55. 10.1186/1742-2094-10-55 PubMed DOI PMC

Dubový P., Hradilová-Svíženská I., Klusáková I., Kokošová V., Brázda V., Joukal M. (2018). Bilateral activation of STAT3 by phosphorylation at the tyrosine-705 (Y705) and serine-727 (S727) positions and its nuclear translocation in primary sensory neurons following unilateral sciatic nerve injury. Histochem. Cell Biol. 150, 37–47. 10.1007/s00418-018-1656-y PubMed DOI

Dubový P., Svízenská I. (1994). Denervated skeletal-muscle stimulates migration of Schwann-cells from the distal stump of transected peripheral-nerve: an in vivo study. Glia 12, 99–107. 10.1002/glia.440120203 PubMed DOI

Duregotti E., Negro S., Scorzeto M., Zornetta I., Dickinson B. C., Chang C. J., et al. . (2015). Mitochondrial alarmins released by degenerating motor axon terminals activate perisynaptic Schwann cells. Proc. Nat. Acad. Sci. U S A 112, E497–E505. 10.1073/pnas.1417108112 PubMed DOI PMC

Edström A., Briggman M., Ekström P. A. R. (1996). Phospholipase A2 activity is required for regeneration of sensory axons in cultured adult sciatic nerves. J. Neurosci. Res. 43, 183–189. 10.1002/(sici)1097-4547(19960115)43:2<183::aid-jnr6>3.0.co;2-c PubMed DOI

Forsman H., Andréasson E., Karlsson J., Boulay F., Rabiet M. J., Dahlgren C. (2012). Structural characterization and inhibitory profile of formyl peptide receptor 2 selective peptides descending from a PIP2-binding domain of gelsolin. J. Immunol. 189, 629–637. 10.4049/jimmunol.1101616 PubMed DOI

Gaudet A. D., Popovich P. G., Ramer M. S. (2011). Wallerian degeneration: gaining perspective on inflammatory events after peripheral nerve injury. J. Neuroinflammation 8:110. 10.1186/1742-2094-8-110 PubMed DOI PMC

Goethals S., Ydens E., Timmerman V., Janssens S. (2010). Toll-Like Receptor expression in the peripheral nerve. Glia 58, 1701–1709. 10.1002/glia.21041 PubMed DOI

Gomez-Sanchez J. A., Pilch K. S., van der Lans M., Fazal S. V., Benito C., Wagstaff L. J., et al. . (2017). After nerve injury, lineage tracing shows that myelin and remak Schwann cells elongate extensively and branch to form repair Schwann cells, which shorten radically on remyelination. J. Neurosci. 37, 9086–9099. 10.1523/JNEUROSCI.1453-17.2017 PubMed DOI PMC

Ho C. F. Y., Ismail N. B., Koh J. K. Z., Gunaseelan S., Low Y. H., Ng Y. K., et al. . (2018). Localisation of formyl-peptide receptor 2 in the rat central nervous system and its role in axonal and dendritic outgrowth. Neurochem. Res. 43, 1587–1598. 10.1007/s11064-018-2573-0 PubMed DOI PMC

Hylden J. L. K., Wilcox G. L. (1980). Intrathecal morphine in mice: a new technique. Eur. J. Pharmacol. 67, 313–316. 10.1016/0014-2999(80)90515-4 PubMed DOI

Karanth S., Yang G., Yeh J., Richardson P. M. (2006). Nature of signals that initiate the immune response during Wallerian degeneration of peripheral nerves. Exp. Neurol. 202, 161–166. 10.1016/j.expneurol.2006.05.024 PubMed DOI

Kato J., Svensson C. I. (2015). “Role of extracellular damage-associated molecular pattern molecules (DAMPs) as mediators of persistent pain,” in Molecular and Cell Biology of Pain, eds Price T. J., Dussor G. (Elsevier: Academic Press Inc.), 251–279. PubMed

Kaul D., Habbel P., Derkow K., Krüger C., Franzoni E., Wulczyn F. G., et al. . (2012). Expression of toll-like receptors in the developing brain. PLoS One 7:e37767. 10.1371/journal.pone.0037767 PubMed DOI PMC

Krysko D. V., Agostinis P., Krysko O., Garg A. D., Bachert C., Lambrecht B. N., et al. . (2011). Emerging role of damage-associated molecular patterns derived from mitochondria in inflammation. Trends Immunol. 32, 157–164. 10.1016/j.it.2011.01.005 PubMed DOI

Lacagnina M. J., Watkins L. R., Grace P. M. (2018). Toll-like receptors and their role in persistent pain. Pharmacol. Ther. 184, 145–158. 10.1016/j.pharmthera.2017.10.006 PubMed DOI PMC

Le Y. Y., Murphy P. M., Wang J. M. (2002). Formyl-peptide receptors revisited. Trends Immunol. 23, 541–548. 10.1016/s1471-4906(02)02316-5 PubMed DOI

Lemieux J. M., Wu G., Morgan J. A., Kacena M. A. (2011). DMSO regulates osteoclast development in vitro. In Vitro Cell. Dev. Biol. Anim. 47, 260–267. 10.1007/s11626-011-9385-8 PubMed DOI PMC

Leventhal P. S., Shelden E. A., Kim B., Feldman E. L. (1997). Tyrosine phosphorylation of paxillin and focal adhesion kinase during insulin-like growth factor-I-stimulated lamellipodial advance. J. Biol. Chem. 272, 5214–5218. 10.1074/jbc.272.8.5214 PubMed DOI

Liu C., Kray J., Toomajian V., Chan C. (2016). Schwann cells migration on patterned polydimethylsiloxane microgrooved surface. Tissue Eng. Part C Methods 22, 644–651. 10.1089/ten.tec.2015.0539 PubMed DOI PMC

López-Colomé A. M., Lee-Rivera I., Benavides-Hidalgo R., López E. (2017). Paxillin: a crossroad in pathological cell migration. J. Hematol. Oncol. 10:50. 10.1186/s13045-017-0418-y PubMed DOI PMC

Miyamoto Y., Torii T., Yamamori N., Eguchi T., Nagao M., Nakamura K., et al. . (2012). Paxillin is the target of c-Jun N-terminal kinase in Schwann cells and regulates migration. Cell. Signal. 24, 2061–2069. 10.1016/j.cellsig.2012.06.013 PubMed DOI

Monlun M., Hyernard C., Blanco P., Lartigue L., Faustin B. (2017). Mitochondria as molecular platforms integrating multiple innate immune signalings. J. Mol. Biol. 429, 1–13. 10.1016/j.jmb.2016.10.028 PubMed DOI

Negro S., Stazi M., Marchioretto M., Tebaldi T., Rodella U., Duregotti E., et al. . (2018). Hydrogen peroxide is a neuronal alarmin that triggers specific RNAs, local translation of Annexin A2, and cytoskeletal remodeling in Schwann cells. RNA 24, 915–925. 10.1261/rna.064816.117 PubMed DOI PMC

Pacheco A., Gallo G. (2016). Actin filament-microtubule interactions in axon initiation and branching. Brain Res. Bull. 126, 300–310. 10.1016/j.brainresbull.2016.07.013 PubMed DOI PMC

Park J. Y., Jang S. Y., Shin Y. K., Koh H., Suh D. J., Shinji T., et al. . (2013). Mitochondrial swelling and microtubule depolymerization are associated with energy depletion in axon degeneration. Neuroscience 238, 258–269. 10.1016/j.neuroscience.2013.02.033 PubMed DOI

Romanova L. Y., Mushinski J. F. (2011). Central role of paxillin phosphorylation in regulation of LFA-1 integrins activity and lymphocyte migration. Cell Adhes. Migr. 5, 457–462. 10.4161/cam.5.6.18219 PubMed DOI PMC

Ronchi G., Raimondo S., Varejão A. S. P., Tos P., Perroteau I., Geuna S. (2010). Standardized crush injury of the mouse median nerve. J. Neurosci. Methods 188, 71–75. 10.1016/j.jneumeth.2010.01.024 PubMed DOI

Scheib J., Höeke A. (2013). Advances in peripheral nerve regeneration. Nat. Rev. Neurol. 9, 668–676. 10.1038/nrneurol.2013.227 PubMed DOI

Son Y. J., Thompson W. J. (1995a). Nerve sprouting in muscle is induced and guided by processes extended by Schwann-cells. Neuron 14, 133–141. 10.1016/0896-6273(95)90247-3 PubMed DOI

Son Y. J., Thompson W. J. (1995b). Schwann-cell processes guide regeneration of peripheral axons. Neuron 14, 125–132. 10.1016/0896-6273(95)90246-5 PubMed DOI

Stoll G., Jander S., Myers R. R. (2002). Degeneration and regeneration of the peripheral nervous system: from Augustus Waller’s observations to neuroinflammation. J. Peripher. Nerv. Syst. 7, 13–27. 10.1046/j.1529-8027.2002.02002.x PubMed DOI

Takeshita F., Gursel I., Ishii K. J., Suzuki K., Gursel M., Klinman D. M. (2004). Signal transduction pathways mediated by the interaction of CpG DNA with Toll-like receptor 9. Sem. Immunol. 16, 17–22. 10.1016/j.smim.2003.10.009 PubMed DOI

VanCompernolle S. E., Clark K. L., Rummel K. A., Todd S. C. (2003). Expression and function of formyl peptide receptors on human fibroblast cells. J. Immunol. 171, 2050–2056. 10.4049/jimmunol.171.4.2050 PubMed DOI

Vindis C., Teli T., Cerretti D. P., Turner C. E., Huynh-Do U. (2004). EphB1-mediated cell migration requires the phosphorylation of paxillin at Tyr-31/Tyr-118. J. Biol. Chem. 279, 27965–27970. 10.1074/jbc.M401295200 PubMed DOI

Walsh D., McCarthy J., O’Driscoll C., Melgar S. (2013). Pattern recognition receptors—molecular orchestrators of inflammation in inflammatory bowel disease. Cytokine Growth Factor Rev. 24, 91–104. 10.1016/j.cytogfr.2012.09.003 PubMed DOI

Wang G., Zhang L., Chen X. X., Xue X., Guo Q. N., Liu M. Y., et al. . (2016). Formylpeptide receptors promote the migration and differentiation of rat neural stem cells. Sci. Rep. 6:25946. 10.1038/srep25946 PubMed DOI PMC

Weiner J. A., Fukushima N., Contos J. J., Scherer S. S., Chun J. (2001). Regulation of Schwann cell morphology and adhesion by receptor-mediated lysophosphatidic acid signaling. J. Neurosci. 21, 7069–7078. 10.1523/JNEUROSCI.21-18-07069.2001 PubMed DOI PMC

Wenceslau C. F., McCarthy C. G., Goulopoulou S., Szasz T., NeSmith E. G., Webb R. C. (2013). Mitochondrial-derived N-formyl peptides: novel links between trauma, vascular collapse and sepsis. Med. Hypoth. 81, 532–535. 10.1016/j.mehy.2013.06.026 PubMed DOI PMC

Xu J., Wang F., Van Keymeulen A., Herzmark P., Straight A., Kelly K., et al. . (2003). Divergent signals and cytoskeletal assemblies regulate self-organizing polarity in neutrophils. Cell 114, 201–214. 10.1016/S0092-8674(03)00555-5 PubMed DOI

Zamboni L., Demartin C. (1967). Buffered picric acid-formaldehyde: a new rapid fixative for electron microscopy. J. Cell Biol. 35:A148.

Zhang Q., Raoof M., Chen Y., Sumi Y., Sursal T., Junger W., et al. . (2010). Circulating mitochondrial DAMPs cause inflammatory responses to injury. Nature 464, 104–115. 10.1038/nature08780 PubMed DOI PMC

Toll-Like Receptor 9-Mediated Neuronal Innate Immune Reaction Is Associated with Initiating a Pro-Regenerative State in Neurons of the Dorsal Root Ganglia Non-Associated with Sciatic Nerve Lesion

N-Formylated Peptide Induces Increased Expression of Both Formyl Peptide Receptor 2 (Fpr2) and Toll-Like Receptor 9 (TLR9) in Schwannoma Cells-An In Vitro Model for Early Inflammatory Profiling of Schwann Cells