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Choline supplementation mitigates effects of bilirubin in cerebellar granule neurons in vitro

Pediatric Research (2024)Cite this article

Abstract

Background

Premature infants may suffer from high levels of bilirubin that could lead to neurotoxicity. Bilirubin has been shown to decrease L1-mediated ERK1/2 signaling, L1 phosphorylation, and L1 tyrosine 1176 dephosphorylation. Furthermore, bilirubin redistributes L1 into lipid rafts (LR) and decreases L1-mediated neurite outgrowth. We demonstrate that choline supplementation improves L1 function and signaling in the presence of bilirubin.

Methods

Cerebellar granule neurons (CGN) were cultured with and without supplemental choline, and the effects on L1 signaling and function were measured in the presence of bilirubin. L1 activation of ERK1/2, L1 phosphorylation and dephosphorylation were measured. L1 distribution in LR was quantified and neurite outgrowth of CGN was determined.

Results

Forty µM choline significantly reduced the effect of bilirubin on L1 activation of ERK1/2 by 220% (p = 0.04), and increased L1 triggered changes in tyrosine phosphorylation /dephosphorylation of L1 by 34% (p = 0.026) and 35% (p = 0.02) respectively. Choline ameliorated the redistribution of L1 in lipid rafts by 38% (p = 0.02) and increased L1-mediated mean neurite length by 11% (p = 0.04).

Conclusion

Choline pretreatment of CGN significantly reduced the disruption of L1 function by bilirubin. The supplementation of pregnant women and preterm infants with choline may increase infant resilience to the effects of bilirubin.

Impact

  • This article establishes choline as an intervention for the neurotoxic effects of bilirubin on lipid rafts.

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

  • This article paves the way for future investigation into the mechanism of the ameliorative effect of choline on bilirubin neurotoxicity.

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Fig. 1: Choline reduces bilirubin inhibition of ERK1/2 activation.
Fig. 2: Choline reduces bilirubin inhibition of L1 tyrosine phosphorylation.
Fig. 3: Choline reduces bilirubin inhibition of L1 dephosphorylation.
Fig. 4: Choline reduces bilirubin-induced L1 redistribution in lipid rafts.
Fig. 5: Choline reduces bilirubin inhibition of neurite outgrowth.
Fig. 6: Neurites visualized with fluorescent microscopy.

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Acknowledgements

This work was supported by NIH R21HD085061, NIH R21HD105071, Mary Gray Cobey Endowment and Munro Funds. The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request. This material is original and has not been previously published nor has it been submitted for publication elsewhere while under consideration.

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Authors and Affiliations

  1. Department of Pediatrics, Case Western Reserve University School of Medicine, Cleveland, OH, 44106, USA

    Mrinaj Janampalli, Spencer T. Kitchen, Sergei Vatolin & Cynthia F. Bearer

  2. Department of Orthopedics, University of Maryland School of Medicine, Baltimore, MD, 21201, USA

    Ningfeng Tang

  3. Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, 21218, USA

    Min He

  4. Division of Neonatology, Department of Pediatrics, UH Rainbow Babies & Children’s Hospital, Cleveland, OH, 44106, USA

    Cynthia F. Bearer

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Contributions

M.J. substantially contributed to the acquisition and analysis of data in this article as well as drafting and revising of the article. S.T.K. substantially contributed to the analysis and interpretation of data, and drafting and revising of this article. S.V. substantially contributed to the analysis and interpretation of data, and drafting and revising of this article. N.T. substantially contributed to the design of this study and the acquisition and analysis of data in this article. M.H. substantially contributed to the design of this study and the acquisition and analysis of data in 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. This study was funded by R21HD085061 and R21HD105071, Rockville, MD (CB).

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Correspondence to Cynthia F. Bearer.

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Janampalli, M., Kitchen, S.T., Vatolin, S. et al. Choline supplementation mitigates effects of bilirubin in cerebellar granule neurons in vitro. Pediatr Res (2024). https://doi.org/10.1038/s41390-023-02968-6

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