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
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This article establishes lipid rafts as a target for the neurotoxic effects of bilirubin.
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This article provides clear evidence toward establishing one mechanism of bilirubin neurotoxicity, where little is understood.
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This article paves the way for future investigation into lipid raft dependent functions, and its role in neurodevelopmental outcome.
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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).
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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.
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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 89, 1389–1395 (2021). https://doi.org/10.1038/s41390-020-01156-0
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DOI: https://doi.org/10.1038/s41390-020-01156-0
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