Lipopolysaccharide and Morphine-3-Glucuronide-Induced Immune Signalling Increases the Expression of Polysialic Acid in PC12 Cells


Polysialic acid (polySia), a long homopolymer of 2,8-linked sialic acids, is abundant in the embryonic brain and is restricted largely in adult brain to regions that exhibit neurogenesis and structural plasticity. In the central nervous system (CNS), polySia is highly important for cell-cell interactions, differentiation, migration and cytokine responses, which are critical neuronal functions regulating intercellular interactions that underlie immune signalling in the CNS. In recent reports, a metabolite of morphine, morphine-3-glucuronide (M3G), has been shown to cause immune signalling in the CNS. In this study, we compared the effects of neurite growth factor (NGF), lipopolysaccharide (LPS) and M3G exposure on the expression of polySia in PC12 cells using immunocytochemistry and Western blot analysis. PolySia was also extracted from stimulated cell proteins by endo-neuraminidase digestion and quantitated using fluorescent labelling followed by HPLC analysis. PolySia expression was significantly increased following NGF, M3G or LPS stimulation when compared with unstimulated cells or cells exposed to the TLR4 antagonist LPS-RS. Additionally, we analyzed the effects of test agent exposure on cell migration and the oxidative stress response of these cells in the presence and absence of polySia expression on their cell surface. We observed an increase in oxidative stress in cells without polySia as well as following M3G or LPS stimulation. Our study provides evidence that polySia expression in neuronal-like PC12 cells is influenced by M3G and LPS exposure alike, suggestive of a role of TLR4 in triggering these events.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6



central nervous system


degree of polymerization


Dulbecco’s modified Eagle’s medium






mitogen-activated protein kinase




neurite growth factor


polysialic acid


reactive oxygen species


toll-like receptor 4


tumour necrosis factor-α


  1. 1.

    Yu RK, Schengrund C-L (2014) Glycobiology of the nervous system. Springer.

    Google Scholar 

  2. 2.

    Angata T, Varki A (2002) Chemical diversity in the sialic acids and related alpha-keto acids: an evolutionary perspective. Chem Rev 102(2):439–469

    CAS  Article  Google Scholar 

  3. 3.

    Schnaar RL, Gerardy-Schahn R, Hildebrandt H (2014) Sialic acids in the brain: gangliosides and polysialic acid in nervous system development, stability, disease, and regeneration. Physiol Rev 94(2):461–518.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Inoue S, Inoue Y (2001) Developmental profile of neural cell adhesion molecule glycoforms with a varying degree of polymerization of polysialic acid chains. J Biol Chem 276(34):31863–31870.

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Nakata D, Troy FA 2nd (2005) Degree of polymerization (DP) of polysialic acid (polySia) on neural cell adhesion molecules (N-CAMS): development and application of a new strategy to accurately determine the DP of polySia chains on N-CAMS. J Biol Chem 280(46):38305–38316.

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    Sato C, Kitajima K (2013) Disialic, oligosialic and polysialic acids: distribution, functions and related disease. J Biochem 154(2):115–136.

    CAS  Article  PubMed  Google Scholar 

  7. 7.

    Bonfanti L (2006) PSA-NCAM in mammalian structural plasticity and neurogenesis. Prog Neurobiol 80(3):129–164.

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Colley KJ, Kitajima K, Sato C (2014) Polysialic acid: biosynthesis, novel functions and applications. Crit Rev Biochem Mol Biol 49(6):498–532.

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Rutishauser U (2008) Polysialic acid in the plasticity of the developing and adult vertebrate nervous system. Nat Rev Neurosci 9(1):26–35.

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Zhang H, Vutskits L, Calaora V, Durbec P, Kiss JZ (2004) A role for the polysialic acid-neural cell adhesion molecule in PDGF-induced chemotaxis of oligodendrocyte precursor cells. J Cell Sci 117(Pt 1):93–103.

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Petridis AK, El-Maarouf A, Rutishauser U (2004) Polysialic acid regulates cell contact-dependent neuronal differentiation of progenitor cells from the subventricular zone. Dev Dyn 230(4):675–684.

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Covacu R, Brundin L (2015) Effects of Neuroinflammation on neural stem cells. Neuroscientist.

    Article  Google Scholar 

  13. 13.

    Sandkühler J (2017) Neuroinflammation and neuroplasticity in pain. Oxford University Press.

  14. 14.

    Jin K, Wang X, Xie L, Mao XO, Zhu W, Wang Y, Shen J, Mao Y et al (2006) Evidence for stroke-induced neurogenesis in the human brain. Proc Natl Acad Sci U S A 103(35):13198–13202.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Tomassini V, d'Ambrosio A, Petsas N, Wise RG, Sbardella E, Allen M, Tona F, Fanelli F et al (2016) The effect of inflammation and its reduction on brain plasticity in multiple sclerosis: MRI evidence. Hum Brain Mapp 37(7):2431–2445.

    Article  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Radley J, Morilak D, Viau V, Campeau S (2015) Chronic stress and brain plasticity: Mechanisms underlying adaptive and maladaptive changes and implications for stress-related CNS disorders. Neurosci Biobehav Rev 58:79–91.

    Article  PubMed  PubMed Central  Google Scholar 

  17. 17.

    El Maarouf A, Kolesnikov Y, Pasternak G, Rutishauser U (2005) Polysialic acid-induced plasticity reduces neuropathic insult to the central nervous system. Proc Natl Acad Sci U S A 102(32):11516–11520.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Dauphinee SM, Karsan A (2006) Lipopolysaccharide signaling in endothelial cells. Lab Invest 86(1):9–22.

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Werneburg S, Buettner FF, Erben L, Mathews M, Neumann H, Muhlenhoff M, Hildebrandt H (2016) Polysialylation and lipopolysaccharide-induced shedding of E-selectin ligand-1 and neuropilin-2 by microglia and THP-1 macrophages. Glia 64(8):1314–1330.

    Article  PubMed  Google Scholar 

  20. 20.

    Ferraz CC, Henry MA, Hargreaves KM, Diogenes A (2011) Lipopolysaccharide from Porphyromonas gingivalis sensitizes capsaicin-sensitive nociceptors. J Endod 37(1):45–48.

    Article  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Milligan ED, Watkins LR (2009) Pathological and protective roles of glia in chronic pain. Nat Rev Neurosci 10(1):23–36.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Wang X, Loram LC, Ramos K, de Jesus AJ, Thomas J, Cheng K, Reddy A, Somogyi AA et al (2012) Morphine activates neuroinflammation in a manner parallel to endotoxin. Proc Natl Acad Sci U S A 109(16):6325–6330.

    Article  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Lewis SS, Hutchinson MR, Rezvani N, Loram LC, Zhang Y, Maier SF, Rice KC, Watkins LR (2010) Evidence that intrathecal morphine-3-glucuronide may cause pain enhancement via toll-like receptor 4/MD-2 and interleukin-1beta. Neuroscience 165(2):569–583.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Hewett K, Dickenson AH, McQuay HJ (1993) Lack of effect of morphine-3-glucuronide on the spinal antinociceptive actions of morphine in the rat: an electrophysiological study. Pain 53(1):59–63

    CAS  Article  Google Scholar 

  25. 25.

    Bartlett SE, Cramond T, Smith MT (1994) The excitatory effects of morphine-3-glucuronide are attenuated by LY274614, a competitive NMDA receptor antagonist, and by midazolam, an agonist at the benzodiazepine site on the GABAA receptor complex. Life Sci 54(10):687–694

    CAS  Article  Google Scholar 

  26. 26.

    Woolf CJ (1981) Intrathecal high dose morphine produces hyperalgesia in the rat. Brain Res 209(2):491–495

    CAS  Article  Google Scholar 

  27. 27.

    Gong QL, Hedner J, Bjorkman R, Hedner T (1992) Morphine-3-glucuronide may functionally antagonize morphine-6-glucuronide induced antinociception and ventilatory depression in the rat. Pain 48(2):249–255

    CAS  Article  Google Scholar 

  28. 28.

    Hutchinson MR, Zhang Y, Shridhar M, Evans JH, Buchanan MM, Zhao TX, Slivka PF, Coats BD et al (2010) Evidence that opioids may have toll-like receptor 4 and MD-2 effects. Brain Behav Immun 24(1):83–95.

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Due MR, Piekarz AD, Wilson N, Feldman P, Ripsch MS, Chavez S, Yin H, Khanna R et al (2012) Neuroexcitatory effects of morphine-3-glucuronide are dependent on Toll-like receptor 4 signaling. J Neuroinflammation 9:200.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Hallenbeck PC, Vimr ER, Yu F, Bassler B, Troy FA (1987) Purification and properties of a bacteriophage-induced endo-N-acetylneuraminidase specific for poly-alpha-2,8-sialosyl carbohydrate units. J Biol Chem 262(8):3553–3561

    CAS  PubMed  Google Scholar 

  31. 31.

    Jensen EC (2013) Quantitative analysis of histological staining and fluorescence using ImageJ. Anat Rec (Hoboken) 296(3):378–381.

    Article  Google Scholar 

  32. 32.

    Varki A, Diaz S (1984) The release and purification of sialic acids from glycoconjugates: methods to minimize the loss and migration of O-acetyl groups. Anal Biochem 137(1):236–247

    CAS  Article  Google Scholar 

  33. 33.

    Liang CC, Park AY, Guan JL (2007) In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc 2(2):329–333.

    CAS  Article  PubMed  Google Scholar 

  34. 34.

    Al-Saraireh YM, Sutherland M, Springett BR, Freiberger F, Ribeiro Morais G, Loadman PM, Errington RJ, Smith PJ et al (2013) Pharmacological inhibition of polysialyltransferase ST8SiaII modulates tumour cell migration. PLoS One 8(8):e73366.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Sato C, K Kitajima (2008) Structural analysis of polysialic acid. Experimental Glycoscience in Taniguchi N, Suzuki A, Ito Y, Narimatsu H, Kawasaki T, Hase S (eds): 77–81.

  36. 36.

    Gaikwad S, Agrawal-Rajput R (2015) Lipopolysaccharide from Rhodobacter sphaeroides attenuates microglia-mediated inflammation and phagocytosis and directs regulatory T cell response. Int J Inflam 2015:361326.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Kirikae T, Schade FU, Kirikae F, Qureshi N, Takayama K, Rietschel ET (1994) Diphosphoryl lipid A derived from the lipopolysaccharide (LPS) of Rhodobacter sphaeroides ATCC 17023 is a potent competitive LPS inhibitor in murine macrophage-like J774.1 cells. FEMS Immunol Med Microbiol 9(3):237–243

    CAS  Article  Google Scholar 

  38. 38.

    Greene LA, Tischler AS (1976) Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci U S A 73(7):2424–2428

    CAS  Article  Google Scholar 

  39. 39.

    Kanato Y, Kitajima K, Sato C (2008) Direct binding of polysialic acid to a brain-derived neurotrophic factor depends on the degree of polymerization. Glycobiology 18(12):1044–1053.

    CAS  Article  PubMed  Google Scholar 

  40. 40.

    Sierra-Fonseca JA, Najera O, Martinez-Jurado J, Walker EM, Varela-Ramirez A, Khan AM, Miranda M, Lamango NS et al (2014) Nerve growth factor induces neurite outgrowth of PC12 cells by promoting Gbetagamma-microtubule interaction. BMC Neurosci 15:132–119.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Tian X, Yue R, Zeng H, Li H, Shan L, He W, Shen Y, Zhang W (2015) Distinctive effect on nerve growth factor-induced PC12 cell neurite outgrowth by two unique neolignan enantiomers from Illicium merrillianum. Sci Rep 5:16982.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Wigerius M, Asghar N, Melik W, Johansson M (2013) Scribble controls NGF-mediated neurite outgrowth in PC12 cells. Eur J Cell Biol 92(6-7):213–221.

    CAS  Article  PubMed  Google Scholar 

  43. 43.

    Greene LA (1978) Nerve growth factor prevents the death and stimulates the neuronal differentiation of clonal PC12 pheochromocytoma cells in serum-free medium. J Cell Biol 78(3):747–755

    CAS  Article  Google Scholar 

  44. 44.

    Angata K, Huckaby V, Ranscht B, Terskikh A, Marth JD, Fukuda M (2007) Polysialic acid-directed migration and differentiation of neural precursors are essential for mouse brain development. Mol Cell Biol 27(19):6659–6668.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Aruoma OI, Grootveld M, Bahorun T (2006) Free radicals in biology and medicine: from inflammation to biotechnology. Biofactors 27(1-4):1–3

    CAS  Article  Google Scholar 

  46. 46.

    Halliwell B, Whiteman M (2004) Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean? Br J Pharmacol 142(2):231–255.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Newton K, Dixit VM (2012) Signaling in innate immunity and inflammation. Cold Spring Harb Perspect Biol 4(3).

    Article  Google Scholar 

  48. 48.

    Varki A (2011) Since there are PAMPs and DAMPs, there must be SAMPs? Glycan "self-associated molecular patterns" dampen innate immunity, but pathogens can mimic them. Glycobiology 21(9):1121–1124

    CAS  Article  Google Scholar 

  49. 49.

    Ramos-Martinez I, Martinez-Loustalot P, Lozano L, Issad T, Limon D, Diaz A, Perez-Torres A, Guevara J et al (2018) Neuroinflammation induced by amyloid beta25-35 modifies mucin-type O-glycosylation in the rat's hippocampus. Neuropeptides 67:56–62.

    CAS  Article  PubMed  Google Scholar 

  50. 50.

    Starossom SC, Mascanfroni ID, Imitola J, Cao L, Raddassi K, Hernandez SF, Bassil R, Croci DO et al (2012) Galectin-1 deactivates classically activated microglia and protects from inflammation-induced neurodegeneration. Immunity 37(2):249–263.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Biancheri R, Falace A, Tessa A, Pedemonte M, Scapolan S, Cassandrini D, Aiello C, Rossi A et al (2007) POMT2 gene mutation in limb-girdle muscular dystrophy with inflammatory changes. Biochem Biophys Res Commun 363(4):1033–1037.

    CAS  Article  PubMed  Google Scholar 

  52. 52.

    Sato S, St-Pierre C, Bhaumik P, Nieminen J (2009) Galectins in innate immunity: dual functions of host soluble beta-galactoside-binding lectins as damage-associated molecular patterns (DAMPs) and as receptors for pathogen-associated molecular patterns (PAMPs). Immunol Rev 230(1):172–187.

    CAS  Article  PubMed  Google Scholar 

  53. 53.

    Davicino RC, Elicabe RJ, Di Genaro MS, Rabinovich GA (2011) Coupling pathogen recognition to innate immunity through glycan-dependent mechanisms. Int Immunopharmacol 11(10):1457–1463.

    CAS  Article  PubMed  Google Scholar 

  54. 54.

    Rutishauser U, Acheson A, Hall AK, Mann DM, Sunshine J (1988) The neural cell adhesion molecule (NCAM) as a regulator of cell-cell interactions. Science 240(4848):53–57

    CAS  Article  Google Scholar 

  55. 55.

    Rutishauser U, Landmesser L (1996) Polysialic acid in the vertebrate nervous system: a promoter of plasticity in cell-cell interactions. Trends Neurosci 19(10):422–427

    CAS  Article  Google Scholar 

  56. 56.

    Seki T, Arai Y (1993) Distribution and possible roles of the highly polysialylated neural cell adhesion molecule (NCAM-H) in the developing and adult central nervous system. Neurosci Res 17(4):265–290

    CAS  Article  Google Scholar 

  57. 57.

    El Maarouf A, Petridis AK, Rutishauser U (2006) Use of polysialic acid in repair of the central nervous system. Proc Natl Acad Sci U S A 103(45):16989–16994.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Leow-Dyke S, Allen C, Denes A, Nilsson O, Maysami S, Bowie AG, Rothwell NJ, Pinteaux E (2012) Neuronal Toll-like receptor 4 signaling induces brain endothelial activation and neutrophil transmigration in vitro. J Neuroinflammation 9:230.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Iqbal S, Ghanimi Fard M, Everest-Dass A, Packer NH, Parker LM (2019) Understanding cellular glycan surfaces in the central nervous system. Biochem Soc Trans 47(1):89–100.

    CAS  Article  PubMed  Google Scholar 

  60. 60.

    Aktas O, Ullrich O, Infante-Duarte C, Nitsch R, Zipp F (2007) Neuronal damage in brain inflammation. Arch Neurol 64(2):185–189.

    Article  PubMed  Google Scholar 

  61. 61.

    Martich GD, Boujoukos AJ, Suffredini AF (1993) Response of man to endotoxin. Immunobiology 187(3-5):403–416.

    CAS  Article  PubMed  Google Scholar 

  62. 62.

    Wolff SM, Rubenstein M, Mulholland JH, Alling DW (1965) Comparison of hematologic and febrile response to endotoxin in man. Blood 26:190–201

    CAS  Article  Google Scholar 

  63. 63.

    Hemstapat K, Monteith GR, Smith D, Smith MT (2003) Morphine-3-glucuronide's neuro-excitatory effects are mediated via indirect activation of N-methyl-D-aspartic acid receptors: mechanistic studies in embryonic cultured hippocampal neurones. Anesth Analg 97(2):494–505 table of contents

    CAS  Article  Google Scholar 

  64. 64.

    Olmos G, Llado J (2014) Tumor necrosis factor alpha: a link between neuroinflammation and excitotoxicity. Mediators Inflamm 2014:861231.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  65. 65.

    Wang X, Michaelis EK (2010) Selective neuronal vulnerability to oxidative stress in the brain. Front Aging Neurosci 2:12.

    Article  PubMed  PubMed Central  Google Scholar 

  66. 66.

    Fischer R, Maier O (2015) Interrelation of oxidative stress and inflammation in neurodegenerative disease: role of TNF. Oxid Med Cell Longev 2015:610813.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  67. 67.

    Shahraz A, Kopatz J, Mathy R, Kappler J, Winter D, Kapoor S, Schutza V, Scheper T et al (2015) Anti-inflammatory activity of low molecular weight polysialic acid on human macrophages. Sci Rep 5:16800.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  68. 68.

    Ghezzi P (2011) Role of glutathione in immunity and inflammation in the lung. Int J Gen Med 4:105–113.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  69. 69.

    Ogasawara Y, Namai T, Yoshino F, Lee MC, Ishii K (2007) Sialic acid is an essential moiety of mucin as a hydroxyl radical scavenger. FEBS Lett 581(13):2473–2477.

    CAS  Article  PubMed  Google Scholar 

  70. 70.

    Iijima R, Takahashi H, Namme R, Ikegami S, Yamazaki M (2004) Novel biological function of sialic acid (N-acetylneuraminic acid) as a hydrogen peroxide scavenger. FEBS Lett 561(1-3):163–166.

    CAS  Article  PubMed  Google Scholar 

Download references


The authors would like to thank the microscopy unit at Macquarie University for their support and use of equipment facilities. We thank Zeiss Australia for use of the LSM800 confocal microscope. We would also like to thank Prof Rita Gerardy-Schahn for generously providing mAb 735 and Endo-N.

Author Contribution Statement

SI, MH and NP conceived and designed the study. SI conducted all the experiments in this study. LP contributed to the experimental design for stimulating inflammation and data analysis for immunofluorescence. EM, AD and NS contributed to HPLC experimental design and interpretation and reviewing the manuscript. SI prepared and wrote the manuscript. MH, LP and NP critically reviewed and revised the manuscript.


This work is primarily supported by the Australian Research Council (ARC) Centre of Excellence Scheme through the Centre of Excellence for Nanoscale BioPhotonics (CE140100003). LMP is also supported by an Australian Research Council Discovery Early Career Research Award (project number DE180100206).

Author information



Corresponding author

Correspondence to Nicolle H. Packer.

Ethics declarations

Conflict of Interest

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic Supplementary Material


(DOCX 194 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Iqbal, S., Parker, L.M., Everest-Dass, A.V. et al. Lipopolysaccharide and Morphine-3-Glucuronide-Induced Immune Signalling Increases the Expression of Polysialic Acid in PC12 Cells. Mol Neurobiol 57, 964–975 (2020).

Download citation


  • Polysialic acid
  • Sialic acids
  • Glycans
  • Lipopolysaccharide (LPS)
  • Morphine-3-glucuronide (M3G)
  • Inflammation
  • Opioid
  • Central nervous system