Bilirubin inhibits lipid raft dependent functions of L1 cell adhesion molecule in rat pup cerebellar granule neurons

Abstract

Background

The mechanism of bilirubin neurotoxicity is poorly understood. We hypothesize that bilirubin inhibits the function of lipid rafts (LR), microdomains of the plasma membrane critical for signal transduction. To test this hypothesis, we measured the effect of free bilirubin (Bf) between 7.6 and 122.5 nM on LR-dependent functions of L1 cell adhesion molecule (L1).

Methods

Cerebellar granule neurons (CGN) were plated on poly-l-lysine overnight, and neurite length was determined after 1 h treatment with L1 alone or L1 and bilirubin. L1 activation of ERK1/2 was measured in CGN in the presence or absence of bilirubin. The effect of bilirubin on L1 distribution in LR was quantitated, and the localization of bilirubin to LR was determined.

Results

The addition of bilirubin to CGN treated with L1 significantly decreased neurite length compared to L1 alone. L1 activation of ERK1/2 was inhibited by bilirubin. Bilirubin redistributed L1 into LR. Bilirubin was associated only with LR-containing fractions of a sucrose density gradient.

Conclusion

Bf significantly inhibits LR-dependent functions of L1 and are found only associated with LR, suggesting one mechanism by which bilirubin may exert neurotoxicity is through the dysfunction of protein–LR interactions.

Impact

  • This article establishes lipid rafts as a target for the neurotoxic effects of bilirubin.

  • This article provides clear evidence toward establishing one mechanism of bilirubin neurotoxicity, where little is understood.

  • This article paves the way for future investigation into lipid raft dependent functions, and its role in neurodevelopmental outcome.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Bilirubin inhibits L1-mediated neurite outgrowth at physiologic concentrations of Bf.
Fig. 2: Bilirubin does not inhibit laminin-mediated neurite outgrowth at physiologic concentrations of Bf.
Fig. 3: L1 activation of ERK1/2 is dependent on lipid rafts.
Fig. 4: Physiologic bilirubin concentrations inhibit L1 activation of ERK1/2.
Fig. 5: The fraction of total L1 in the lipid raft pool is increased by bilirubin.
Fig. 6: Bilirubin is found only in lipid raft-containing fractions of a sucrose density gradient.

References

  1. 1.

    Shapiro, S. M. & Nakamura, H. Bilirubin and the auditory system. J. Perinatol. 21(Suppl. 1), S52–S55 (2001). Discussion S59–S62.

    PubMed  Google Scholar 

  2. 2.

    Arnold, C., Pedroza, C. & Tyson, J. E. Phototherapy in ELBW newborns: does it work? Is it safe? The evidence from randomized clinical trials. Semin. Perinatol. 38, 452–464 (2014).

    PubMed  Google Scholar 

  3. 3.

    Xoinis, K. et al. Extremely low birth weight infants are at high risk for auditory neuropathy. J. Perinatol. 27, 718–723 (2007).

    CAS  PubMed  Google Scholar 

  4. 4.

    Biran, V., Verney, C. & Ferriero, D. M. Perinatal cerebellar injury in human and animal models. Neurol. Res. Int. 2012, 858929 (2012).

    PubMed  PubMed Central  Google Scholar 

  5. 5.

    Bhutani, V. K., Wong, R. J. & Stevenson, D. K. Hyperbilirubinemia in preterm neonates. Clin. Perinatol. 43, 215–232 (2016).

    PubMed  Google Scholar 

  6. 6.

    Amin, S. B. & Lamola, A. A. Newborn jaundice technologies: unbound bilirubin and bilirubin binding capacity in neonates. Semin. Perinatol. 35, 134–140 (2011).

    PubMed  PubMed Central  Google Scholar 

  7. 7.

    Malik, S. G., Irwanto, K. A., Ostrow, J. D. & Tiribelli, C. Effect of bilirubin on cytochrome c oxidase activity of mitochondria from mouse brain and liver. BMC Res. Notes 3, 162 (2010).

    PubMed  PubMed Central  Google Scholar 

  8. 8.

    Ahlfors, C. E. Predicting bilirubin neurotoxicity in jaundiced newborns. Curr. Opin. Pediatr. 22, 129–133 (2010).

    PubMed  Google Scholar 

  9. 9.

    Shapiro, S. M. Definition of the clinical spectrum of kernicterus and bilirubin-induced neurologic dysfunction (BIND). J. Perinatol. 25, 54–59 (2005).

    CAS  PubMed  Google Scholar 

  10. 10.

    Ostrow, J. D., Pascolo, L. & Tiribelli, C. Reassessment of the unbound concentrations of unconjugated bilirubin in relation to neurotoxicity in vitro. Pediatr. Res. 54, 98–104 (2003).

    CAS  PubMed  Google Scholar 

  11. 11.

    Danbolt, C. et al. In vitro binding of [3H]bilirubin to neurons in rat brain sections. Biol. Neonate 63, 35–39 (1993).

    CAS  PubMed  Google Scholar 

  12. 12.

    Ostrow, J. D., Pascolo, L., Brites, D. & Tiribelli, C. Molecular basis of bilirubin-induced neurotoxicity. Trends Mol. Med. 10, 65–70 (2004).

    CAS  PubMed  Google Scholar 

  13. 13.

    Mukerjee, P. & Ostrow, J. D. Interactions of unconjugated bilirubin with vesicles, cyclodextrins and micelles: new modeling and the role of high pKa values. BMC Biochem. 11, 15 (2010).

    PubMed  PubMed Central  Google Scholar 

  14. 14.

    Simons, K. & Gerl, M. J. Revitalizing membrane rafts: new tools and insights. Nat. Rev. Cell Biol. 11, 688–699 (2010).

    CAS  Google Scholar 

  15. 15.

    Patel, H. H., Murray, F. & Insel, P. A. Caveolae as organizers of pharmacologically relevant signal transduction molecules. Annu. Rev. Pharm. Toxicol. 48, 359–391 (2008).

    CAS  Google Scholar 

  16. 16.

    Epshtein, Y. et al. Identification of a C-terminus domain critical for the sensitivity of Kir2.1 to cholesterol. Proc. Natl Acad. Sci. USA 106, 8055–8060 (2009).

    CAS  PubMed  Google Scholar 

  17. 17.

    Fantini, J. & Barrantes, F. J. Sphingolipid/cholesterol regulation of neurotransmitter receptor conformation and function. Biochim. Biophys. Acta 1788, 2345–2361 (2009).

    CAS  PubMed  Google Scholar 

  18. 18.

    Simons, K. & Ikonen, E. Functional rafts in cell membranes. Nature 387, 569–572 (1997).

    CAS  PubMed  Google Scholar 

  19. 19.

    Dart, C. Lipid microdomains and the regulation of ion channel function. J. Physiol. 588, 3169–3178 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Simons, K. & Ehehalt, R. Cholesterol, lipid rafts, and disease. J. Clin. Invest. 110, 597–603 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Owen, D. M., Magenau, A., Williamson, D. & Gaus, K. The lipid raft hypothesis revisited-new insights on raft composition and function from super-resolution fluorescence microscopy. Bioessays 34, 739–747 (2012).

    CAS  PubMed  Google Scholar 

  22. 22.

    Korade, Z. & Kenworthy, A. K. Lipid rafts, cholesterol, and the brain. Neuropharmacology 55, 1265–1273 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Pike, L. J. The challenge of lipid rafts. J. Lipid Res. 50(Suppl.), S323–S328 (2009).

    PubMed  PubMed Central  Google Scholar 

  24. 24.

    Nakai, Y. & Kamiguchi, H. Migration of nerve growth cones requires detergent-resistant membranes in a spatially defined and substrate-dependent manner. J. Cell Biol. 159, 1097–1108 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Tang, N. et al. Ethanol causes the redistribution of L1 cell adhesion molecule in lipid rafts. J. Neurochem. 119, 859–867 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Kamiguchi, H. & Lemmon, V. Recycling of the cell adhesion molecule L1 in axonal growth cones. J. Neurosci. 20, 3676–3686 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Schmid, R. S., Pruitt, W. M. & Maness, P. F. A. MAP kinase-signaling pathway mediates neurite outgrowth on L1 and requires Src-dependent endocytosis. J. Neurosci. 20, 4177–4188 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Yeaney, N. K. et al. Ethanol inhibits L1 cell adhesion molecule tyrosine phosphorylation and dephosphorylation and activation of pp60(src). J. Neurochem. 110, 779–790 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Watanabe, H. et al. Phospholipase D2 functions as a downstream signaling molecule of MAP kinase pathway in L1-stimulated neurite outgrowth of cerebellar granule neurons. J. Neurochem. 89, 142–151 (2004).

    CAS  PubMed  Google Scholar 

  30. 30.

    Milstone, A. M. et al. Chlorhexidine inhibits L1 cell adhesion molecule-mediated neurite outgrowth in vitro. Pediatr. Res. 75, 8–13 (2014).

    CAS  PubMed  Google Scholar 

  31. 31.

    Tang, N. et al. Choline partially prevents the impact of ethanol on the lipid raft dependent functions of l1 cell adhesion molecule. Alcohol Clin. Exp. Res. 38, 2722–2730 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Gonzalez-Reyes, S. et al. Neuroprotective role of heme-oxygenase 1 against iodoacetate-induced toxicity in rat cerebellar granule neurons: Role of bilirubin. Free Radic. Res. 43, 214–223 (2009).

    CAS  PubMed  Google Scholar 

  33. 33.

    Ahlfors, C. E. Measurement of plasma unbound unconjugated bilirubin. Anal. Biochem. 279, 130–135 (2000).

    CAS  PubMed  Google Scholar 

  34. 34.

    Shapiro, S. M., Sombati, S., Geiger, A. & Rice, A. C. NMDA channel antagonist MK-801 does not protect against bilirubin neurotoxicity. Neonatology 92, 248–257 (2007).

    CAS  PubMed  Google Scholar 

  35. 35.

    Bearer, C. F., Swick, A. R., O’Riordan, M. A. & Cheng, G. Ethanol inhibits L1-mediated neurite outgrowth in postnatal rat cerebellar granule cells. J. Biol. Chem. 274, 13264–13270 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Tang, N. et al. Ethanol inhibits L1 cell adhesion molecule activation of mitogen-activated protein kinases. J. Neurochem. 96, 1480–1490 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Mustafa, M. G. & King, T. E. Binding of bilirubin with lipid. A possible mechanism of its toxic reactions in mitochondria. J. Biol. Chem. 245, 1084–1089 (1970).

    CAS  PubMed  Google Scholar 

  38. 38.

    Diamond, I. & Schmid, R. Oxidative phosphorylation in experimental bilirubin encephalopathy. Science 155, 1288–1289 (1967).

    CAS  PubMed  Google Scholar 

  39. 39.

    Daood, M. J., Hoyson, M. & Watchko, J. F. Lipid peroxidation is not the primary mechanism of bilirubin-induced neurologic dysfunction in jaundiced Gunn rat pups. Pediatr. Res. 72, 455–459 (2012).

    CAS  PubMed  Google Scholar 

  40. 40.

    Nakai, Y. & Kamiguchi, H. Migration of nerve growth cones requires detergent-resistant membranes in a spatially defined and substrate-dependent manner. J. Cell Biol. 159, 1097–1108 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Šmíd, V. et al. Heme oxygenase-1 may affect cell signalling via modulation of ganglioside composition. Oxid. Med. Cell Longev. 2018, 3845027 (2018).

    PubMed  PubMed Central  Google Scholar 

  42. 42.

    Mancuso, C. et al. Bilirubin as an endogenous modulator of neurotrophin redox signaling. J. Neurosci. Res. 86, 2235–2249 (2008).

    CAS  PubMed  Google Scholar 

  43. 43.

    Fernandes, A. et al. MAPKs are key players in mediating cytokine release and cell death induced by unconjugated bilirubin in cultured rat cortical astrocytes. Eur. J. Neurosci. 25, 1058–1068 (2007).

    PubMed  Google Scholar 

  44. 44.

    Peyton, K. J. et al. Bilirubin inhibits neointima formation and vascular smooth muscle cell proliferation and migration. Front. Pharmacol. 3, 48 (2012).

    PubMed  PubMed Central  Google Scholar 

  45. 45.

    Öllinger, R. et al. Bilirubin inhibits tumor cell growth via activation of ERK. Cell Cycle 6, 3078–3085 (2007).

    PubMed  Google Scholar 

  46. 46.

    Lee, Y. K. et al. The significance of measurement of serum unbound bilirubin concentrations in high-risk infants. Pediatr. Int. 51, 795–799 (2009).

    CAS  PubMed  Google Scholar 

  47. 47.

    van der Schoor, L. W. et al. Unconjugated free bilirubin in preterm infants. Early Hum. Dev. 106–107, 25–32 (2017).

    PubMed  Google Scholar 

  48. 48.

    Hulzebos, C. V. et al. The bilirubin albumin ratio in the management of hyperbilirubinemia in preterm infants to improve neurodevelopmental outcome: a randomized controlled trial-BARTrial. PLoS ONE 9, e99466 (2014).

    PubMed  PubMed Central  Google Scholar 

  49. 49.

    Amin, S. B. & Wang, H. Bilirubin albumin binding and unbound unconjugated hyperbilirubinemia in premature infants. J. Pediatr. 192, 47–52 (2018).

    CAS  PubMed  Google Scholar 

  50. 50.

    Bearer, C. F. et al. Choline ameliorates deficits in balance caused by acute neonatal ethanol exposure. Cerebellum 14, 413–420 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Davis, N. L. et al. Choline ameliorates ethanol induced alterations in tyrosine phosphorylation and distribution in detergent-resistant membrane microdomains of L1 cell adhesion molecule in vivo. Birth Defects Res. 112, 480–489 (2020).

    CAS  PubMed  Google Scholar 

  52. 52.

    Monk, B. R., Leslie, F. M. & Thomas, J. D. The effects of perinatal choline supplementation on hippocampal cholinergic development in rats exposed to alcohol during the brain growth spurt. Hippocampus 22, 1750–1757 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Thomas, J. D., Idrus, N. M., Monk, B. R. & Dominguez, H. D. Prenatal choline supplementation mitigates behavioral alterations associated with prenatal alcohol exposure in rats. Birth Defects Res. A Clin. Mol. Teratol. 88, 827–837 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Waddell, J. & Mooney, S. M. Choline and working memory training improve cognitive deficits caused by prenatal exposure to ethanol. Nutrients 9, 1080 (2017).

    PubMed Central  Google Scholar 

  55. 55.

    Zeisel, S. H. Nutrition in pregnancy: the argument for including a source of choline. Int J. Women’s Health 5, 193–199 (2013).

    CAS  Google Scholar 

  56. 56.

    Schneider, J. S. et al. Parkinson’s disease: improved function with GM1 ganglioside treatment in a randomized placebo-controlled study. Neurology 50, 1630–1636 (1998).

    CAS  PubMed  Google Scholar 

  57. 57.

    Kalpoyiannis, N. et al. Efficacy of phototherapy and/or exchange transfusions in neonatal jaundice. Clin. Pediatr. 21, 602–606 (1982).

    CAS  Google Scholar 

  58. 58.

    Geiger, A. S., Rice, A. C. & Shapiro, S. M. Minocycline blocks acute bilirubin-induced neurological dysfunction in jaundiced Gunn rats. Neonatology 92, 219–226 (2007).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This material is original and has not been previously published nor has it been submitted for publication elsewhere while under consideration. We would like to acknowledge the special contributions of Sean Sukys, Hemal N. Sempat, and Nicholas Rickman. This study was funded by NIH R01AA016398 and R21HD085061, Rockville, MD (CB) and the Cobey Endowment, Baltimore, MD (CB) and The Munro Fund, Baltimore, MD (CB).

Author information

Affiliations

Authors

Contributions

S.T.K. substantially contributed to the analysis and interpretation of data, and drafting and revising of this article. N.T. and M.H. substantially contributed to the design of this study and the acquisition and analysis of data in this article. E.L. substantially contributed to the drafting of this article and the acquisition of data in this article. S.M.M. substantially contributed to the design of this study and the revising of this article. C.F.B. substantially contributed to the conception and design of this study and has final approval of the version to be published.

Corresponding author

Correspondence to Cynthia F. Bearer.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

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

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kitchen, S.T., Tang, N., He, M. et al. Bilirubin inhibits lipid raft dependent functions of L1 cell adhesion molecule in rat pup cerebellar granule neurons. Pediatr Res (2020). https://doi.org/10.1038/s41390-020-01156-0

Download citation

Search