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

Kitchen, Spencer T; Tang, Ningfeng; He, Min; Ly, Eric; Mooney, Sandra M; Bearer, Cynthia F

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.Fig. 1BILIRUBIN INHIBITS L1-MEDIATED NEURITE OUTGROWTH AT PHYSIOLOGIC CONCENTRATIONS OF BF.: a Distribution curve of neurite lengths without L1, L1, or L1 plus 5, 10, or 50 µM of bilirubin from a representative experiment. b Bar graph of mean ± SEM neurite lengths of CGN grown without L1, L1, or L1 plus 5, 10, or 50 µM of bilirubin normalized to CGN grown on PLL alone. L1 significantly increases neurite length, and all concentrations of bilirubin tested significantly reduce the mean neurite length relative to the L1 alone group. N = 3, ANOVA p < 0.001, post hoc pairwise comparison (Tukey) *p < 0.0001; **p < 0.001; †p < 0.01.Fig. 2BILIRUBIN DOES NOT INHIBIT LAMININ-MEDIATED NEURITE OUTGROWTH AT PHYSIOLOGIC CONCENTRATIONS OF BF.: a Distribution curve of neurite lengths with PLL alone, laminin (Lam), or Lam plus 5, 10, or 50 µM of bilirubin from a representative experiment. b Bar graph of mean ± SEM neurite lengths of CGN grown without Lam, Lam, or Lam plus 5, 10, or 50 µM of bilirubin normalized to CGN grown on PLL alone. N = 3, ANOVA p < 0.01, post hoc pairwise comparison (Tukey), *p < 0.01, NS non-significant.Fig. 3L1 ACTIVATION OF ERK1/2 IS DEPENDENT ON LIPID RAFTS.: a A representative immunoblot of phosphoERK1/2 and total ERK with and without MBCD (M). The experimental groups are as follows: Mouse IgG (C); clASCS4; MBCD added 1 h prior Mouse IgG alone (M); MBCD added 2 h prior to clASCS4 (M + L1). b Bar graph of mean ± SEM of relative densitometric units of phosphoERK1/2 and total ERK, normalized to the control, N = 3, ANOVA p < 0.0001, post hoc pairwise comparison (Tukey) *p < 0.01.Fig. 4PHYSIOLOGIC BILIRUBIN CONCENTRATIONS INHIBIT L1 ACTIVATION OF ERK1/2.: a A representative immunoblot of phosphoERK1/2 with total ERK as a loading control. CGN treated without clASCS4, clASCS4, or clASCS4 plus 5, 10, or 50 µM bilirubin as indicated for 1 h prior to addition of clASCS4. b Bar graph of mean ± SEM of relative densitometric units of clASCS4 activation of ERK1/2 normalized to control. N = 3, ANOVA p < 0.0001, post hoc pairwise comparison (Tukey) *p < 0.01.Fig. 5THE FRACTION OF TOTAL L1 IN THE LIPID RAFT POOL IS INCREASED BY BILIRUBIN.: a Representative immunoblot showing L1 distribution in lipid rafts (LR) and non-lipid rafts (N) in K5 media alone (control), the presence of 25 mM ethanol (EtOH) as a positive control, or 5 µM bilirubin. b Bar graph of %L1 in lipid rafts (LR × 100/(LR + N) (mean ± SEM). N = 3, ANOVA p < 0.01, post hoc pairwise comparison (Tukey) *p < 0.01, **p < 0.05.Fig. 6BILIRUBIN IS FOUND ONLY IN LIPID RAFT-CONTAINING FRACTIONS OF A SUCROSE DENSITY GRADIENT.: a Representative dot blot of sucrose gradients treated with bilirubin and/or MBCD and reactive to CTXB. b Representative dot blot of the same fractions immunoreactive with bilirubin.