Ethanol causes the redistribution of L1 cell adhesion molecule in lipid rafts

Abstract

Fetal alcohol spectrum disorder is estimated to affect 1% of live births. The similarities between children with fetal alcohol syndrome and those with mutations in the gene encoding L1 cell adhesion molecule (L1) implicates L1 as a target of ethanol developmental neurotoxicity. Ethanol specifically inhibits the neurite outgrowth promoting function of L1 at pharmacologic concentrations. Emerging evidence shows that localized disruption of the lipid rafts reduces L1-mediated neurite outgrowth. We hypothesize that ethanol impairment of the association of L1 with lipid rafts is a mechanism underlying ethanol's inhibition of L1-mediated neurite outgrowth. In this study, we examine the effects of ethanol on the association of L1 and lipid rafts. We show that, in vitro, L1 but not N-cadherin shifts into lipid rafts following treatment with 25 mM ethanol. The ethanol concentrations causing this effect are similar to those inhibiting L1-mediated neurite outgrowth. Increasing chain length of the alcohol demonstrates the same cutoff as that previously shown for inhibition of L1-L1 binding. In addition, in cerebellar granule neurons in which lipid rafts are disrupted with methyl-beta-cyclodextrin, the rate of L1-mediated neurite outgrowth on L1-Fc is reduced to background rate and that this background rate is not ethanol sensitive. These data indicate that ethanol may inhibit L1-mediated neurite outgrowth by retarding L1 trafficking through a lipid raft compartment.

Figure 1

(A) Ethanol does not alter the separation of lipid rafts from cell lysates. Immunoblots of two sucrose density gradients, control and EtOH treated. Top panel (Control) is of neurons not exposed to ethanol. Bottom panel (EtOH) is of neurons exposed to 25 mM ethanol for 1 hour prior to lysis. Fractions of the sucrose density gradients were blotted for both cholera toxin subunit B (CTxB) as a marker of lipid raft fractions, and for transferrin receptor (TfR) as a marker of non-lipid raft fractions. (B) Ethanol shifts L1 into lipid rafts while not affecting N-cadherin distribution. CGN incubated with or without 25 mM ethanol (EtOH) for 1 h are separated into lipid raft (LR) and non-lipid raft (N) pools. Proteins from each pool are precipitated, and reconstituted into equal volumes of sample buffer. Equal volumes from each pool are loaded onto 2 lanes, LR and N, and immunoblotted for L1, then stripped and blotted for N-cadherin and GM1 ganglioside using cholera toxin subunit B (CTxB) as a marker for lipid rafts. (C) Densitometric analysis of blots (n = 4). The total pixels in the L1 or N-cadherin bands in both the LR and N lanes is determined for both control and ethanol exposed cultures. The % of cell adhesion molecules (CAM) in the LR is calculated by taking the pixels in the LR band and dividing by the sum of the pixels in the N and LR bands. The mean +/− SD is shown. Statistically significant difference is indicated (*Paired t test, P<0.04).

Figure 2

Confocal images confirm ethanol's effect on colocalization of L1 and GM1 ganglioside. CGN grown on PLL are pretreated with or without 25 mM ethanol for 1 h, then fixed and labeled for L1 (red) and GM1 ganglioside (green). Cells are visualized using a Leica TCS SP2 confocal microscope. Images are merged to visualize overlapping of L1 and lipid rafts (yellow). A) CGN not exposed to ethanol. No yellow signal is seen. B) CGN treated with ethanol. Extensive overlap is seen, as shown by the yellow signal. C) The % of overlap of the red and green channels is measured in at least 20 cells in 3 separate experiments. The mean overlap is calculated for each condition in each experiment. The mean +/− SD of the individual means is shown. Statistically significant difference is indicated (*Paired t test, P<0.02).

Figure 3

Ethanol has no effect on caveolin-1 association with lipid rafts. A) CGN are treated as described, either no addition (Control) or 25 mM ethanol for 1 h (EtOH), prior to preparation of lipid rafts. Lipid rafts (LR) were isolated from non-lipid rafts (N) by sucrose density gradient, pooled, protein precipitated and immunoblotted for caveolin-1. Blots were stripped and reblotted for L1 and CTxB. B) Blots (n=3) were quantified by densitometry. Mean values +/− SD are shown.

Figure 4

Ethanol shifts beta III tubulin out of lipid rafts, opposite to the effect on L1. A) CGN are treated as described in Figure 3. Lipid rafts (LR) were isolated from non-lipid rafts (N) by sucrose density gradient, pooled and western blotted for beta III tubulin. B) Blots were stripped and reblotted for L1. C) Blots from 4 separate experiments quantified by densitometry. Shown are mean values +/− SD. *p<0.05, paired t test.

Figure 5

Butanol redistributes L1 in lipid rafts, but pentanol does not. (A) CGN pretreated with equipotent concentrations of ethanol (EtOH), butanol (BtOH) and pentanol (PtOH) are separated into lipid rafts and non-lipid rafts as described, and immunoblotted for L1. N – Non-lipid raft pool, LR-lipid raft pool. (B) Blots (n=3) are quantified as described in Fig. 1. Mean +/− SD are shown. Statistically significant differences are indicated (*p<0.02, **p<0.003, paired t test).

Figure 6

Ethanol shifts c-Src out of lipid rafts, opposite to the effect on L1. A) CGN are treated as described, either no addition (Control) or 25 mM ethanol for 1 h (EtOH), prior to preparation of lipid rafts. Lipid rafts (LR) were isolated from non-lipid rafts (N) by sucrose density gradient, pooled, protein precipitated and immunoblotted for Src. Blots were stripped and reblotted for L1. B) Blots (n=3) were quantified by densitometry. Mean values +/− SD. *p<0.05, paired t test.

Figure 7

Ethanol shifts GABAA receptor out of lipid rafts, opposite to the effect on L1. A) CGN are treated as described in Figure 5. Lipid rafts (LR) were isolated from non-lipid rafts (N) by sucrose density gradient and western blotted for GABAA receptor beta 2 subunit. B) Blots from 3 separate experiments quantified by densitometry. Shown are mean values +/− SD. *p<0.05, paired t test.

Figure 8

L1 redistribution to lipid rafts is dependent on ethanol concentration. A) CGN are prepared as described and cultured overnight. Ethanol is added at the indicated concentrations for 1 h, then cells are harvested and lipid rafts isolated. Both the lipid raft pool (LR) and non-lipid raft pool (N) are immunoblotted for L1 (L1) and cholera toxin B (CTxB). B) The percent of L1 in lipid rafts (% L1 in LR), calculated as described in the text, are plotted for each concentration of ethanol. Shown are means +/− SD (n=3).

Figure 9

L1 mediated neurite outgrowth is dependent on lipid rafts. A) Immunoblot of L1 in sucrose density gradient fractions of CGN treated with or without MBCD. All detectable L1 colocalizes with the transferrin receptor (TfR), a marker for the non-lipid raft fraction. B) Neurite length of CGN in the presence of various reagents. Cells are plated on either PLL alone (Control) or PLL and L1-Fc (L1-Fc). Ethanol (25 mM) and/or MBCD (4mM) are added 2 h after plating. Cells are fixed 12 h after plating, and neurite length is measured. Shown is mean neurite length +/− SD. Paired t test: a, p<0.0002 from control; b, p< 0.0002 from L1 -Fc; c, p<0.02 from Control; d, p not significant from Control; e, p not significant from L1-Fc+MBCD nor Control.

Figure 9

L1 mediated neurite outgrowth is dependent on lipid rafts. A) Immunoblot of L1 in sucrose density gradient fractions of CGN treated with or without MBCD. All detectable L1 colocalizes with the transferrin receptor (TfR), a marker for the non-lipid raft fraction. B) Neurite length of CGN in the presence of various reagents. Cells are plated on either PLL alone (Control) or PLL and L1-Fc (L1-Fc). Ethanol (25 mM) and/or MBCD (4mM) are added 2 h after plating. Cells are fixed 12 h after plating, and neurite length is measured. Shown is mean neurite length +/− SD. Paired t test: a, p<0.0002 from control; b, p< 0.0002 from L1 -Fc; c, p<0.02 from Control; d, p not significant from Control; e, p not significant from L1-Fc+MBCD nor Control.