Lipid rafts and integrin activation regulate oligodendrocyte survival

Abstract

Newly formed oligodendrocytes in the CNS derive survival cues from their target axons. These cues are provided in part by laminins expressed on the axon, which are recognized by alpha6beta1 integrin on the oligdendrocyte and amplify platelet-derived growth factor (PDGF) signaling through the phosphatidylinositol 3'-kinase (PI3K) pathway. The alpha6beta1 integrin is localized in oligodendrocyte lipid rafts. We show here using the sphingolipid synthesis inhibitor fumonisin-B1 to deplete rafts that this localization is important for normal survival signaling, because depletion increases oligodendrocyte apoptosis and inhibits PI3K signaling. We have shown previously that PDGF-mediated integrin activation is an important component of oligodendrocyte proliferation signaling, and here we present evidence that a similar mechanism operates in survival signaling. Integrin activation using manganese increases raft localization and rescues the effects of both raft depletion and PDGF removal on survival and PI3K signaling. Together, these results point to an essential role for rafts in oligodendrocyte survival signaling on the basis of the provision of a favorable environment for growth factor-mediated integrin activation.

Figure 1.

Effect of raft depletion on PDGFαR and α6β1 localization. A, Phase micrographs of newly differentiated oligodendrocytes, identified by anti-GalC staining, treated with FB1 to deplete lipid rafts, and grown on PDL and Ln-2 substrates. Note the reduction in the number of oligodendrocyte processes in the cells grown in FB1, as quantified in the text. Scale bar, 50 μm. B, Western blots with anti-α6β1 and anti-PDGFαR antibodies of the DIG (raft-containing) and non-DIG (pellet) fractions of Optiprep step gradients prepared as described in Materials and Methods. Note that both the PDGFαR and α6β1 are enriched in the DIG fraction and that this enrichment is greatest on Ln-2 substrates. C, Western blots (PDGFαR) and immunoprecipitations (α6) of sequential fractions (1-bottom, 10-top) of the Optiprep gradients prepared from cells grown with or without FB1 to deplete the rafts. Note that the PDGFαR and the α6β1 are normally enriched in the floating (DIG) fractions that contain insoluble and buoyant raft components (4–6), but after FB1 treatment to deplete rafts, both of the receptors are found in the denser fractions (1–3).

Figure 2.

Oligodendrocyte survival in response to raft depletion and integrin activation. A, The survival assay. Newly differentiated oligodendrocytes grown in culture for 4 d were immunolabeled with anti-GalC (green) and TUNEL (red) with cell nuclei visualized with Hoechst (blue). Apoptotic cells (arrows) were identified as GalC+ (green) and TUNEL+ (red). Scale bar, 25 μm. B, Quantification of survival in increasing concentrations of PDGF in the presence or absence of FB1 or Mn²⁺, or both FB1 and Mn²⁺. Data are presented as the mean percentage change relative to survival on PDL in the absence of PDGF. Note that PDGF-mediated survival is enhanced on Ln-2 compared with PDL and Fn (shown in graphs labeled PDL/FN/LN) and is abolished at lower concentrations of PDGF by the disruption of rafts (graphs labeled +FB1). Mn²⁺ treatment significantly enhanced the survival of oligodendrocytes either in the absence or presence of PDGF, again on all substrates (graphs labeled Mn), and this enhancement is seen even after raft depletion (graphs labeled MN+FB1). Values are means ± SD of at least five independent experiments. Statistical significance was analyzed using the Student's t test. *p < 0.05; **p < 0.02; ***p < 0.01; ****p < 0.001.

Figure 3.

Colocalization of PI3K signaling and lipid rafts in oligodendrocytes. A–D, A fusion protein comprising the Akt pleckstrin-homology domain and GFP (AktPH-GFP) was expressed in oligodendrocytes differentiated on Ln-2 (A, B) and Fn (C, D) substrates (visualized in green). GM1+ rafts were visualized with CTB (red) after paraformaldehyde and methanol fixation. The AktPH-GFP localizes to areas of PI3K signaling (Watton and Downward, 1999) and was expressed in the processes of oligodendrocytes plated on either substrate. Note that cells on Fn have GFP-labeled regions in processes that are unlabeled by CTB (C, D, with merge of bottom arrowed area shown as an inset in D) as well as spatially distinct regions of strong GFP and CTB labeling in the same process (C, D, with top arrowed area shown in inset). In contrast, there is almost complete colocalization of the strongly labeled areas seen in cells on Ln-2 (A, B, arrow). Scale bar: 20 μm. E, Western blotting with anti-pAkt and anti-Akt antibodies of DIG (raft) and non-DIG preparations of cells differentiated in culture on PDL, Ln-2, or Fn for 3–4 d before exposure to PDGF for 30 min. Note the increased relative ratio of pAkt to total Akt within the rafts in the cells grown on Ln-2. F, Western blotting of cells differentiated as above on Ln-2 substrates in the presence of either Ha2/5 anti-β1 integrin Ab (10 μg/ml) or RGD peptides (100 μg/ml) added after 1 d and then exposed to PDGF for 30 min. pAkt is inhibited by the anti-integrin Ab but not the peptide within therafts. Note that no inhibition is seen in the nonraft (non-DIG) compartment, as expected given that the integrin–growth factor receptor interaction occurs within the raft compartment.

Figure 4.

PI3K and MAPK signaling in oligodendrocytes after raft depletion and integrin activation. Serum and growth factor-starved oligodendrocytes differentiated with or without FB1 (to deplete rafts) or Mn²⁺ (to activate integrins) were exposed to PDGF for 30 min. PI3K and MAPK signaling were assessed by Western blotting with antibodies against phosphorylated and total Akt for PI3K (A) and MAPK (B). A, Cells on Ln-2 and Fn showed an increase in Akt phosphorylation (pAkt) as compared with cells on PDL (lane 1), and cells on Ln-2 showed the greatest level of pAkt in response to PDGF (lane 2). Disruption of rafts (+FB1) greatly reduced the level of pAkt on all substrates, most markedly on Ln-2, where levels became undetectable (lane 3). Integrin activation with Mn²⁺ (+Mn) enhanced pAkt levels on all substrates (lane 4) and prevented the reduction seen after raft depletion by FB1 (lane 5). The numbers under each blot are the relative mean intensity of pAkt compared with Akt to normalize the data and facilitate comparison of the different conditions. B, In cells grown on Ln-2, raft depletion induced an increase in the p42/44-MAPK response to PDGF (lane 2) compared with control cells (lane1). This effect was also seen on PDL and Fn substrates (data not shown). Integrin activation with Mn²⁺ induced a large increase in p44-MAPK (lane 3). This level of pMAPK in response to activation was reduced by raft depletion (lane 4) but was still greater than that seen in normal cells (compare lanes 1, 4).

Figure 5.

The effect of integrin activation on α6β1 localization in oligodendrocyte lipid rafts. A, Oligodendrocytes were differentiated on Ln-2 substrates and treated with Mn²⁺ (+Mn) to activate integrins before immunostaining with CTB and anti-α6 antibodies to visualize rafts (green) and the integrin (red). Cells treated with Mn²⁺ expressed more α6β1 within the CTB-labeled areas (arrows) after Mn²⁺ treatment (+Mn) compared with controls (–Mn). Scale bar, 10 μm. B, Immunoprecipitations with anti-α6 or anti-αvβ3 antibodies of DIG and non-DIG preparations containing the same quantities of protein and prepared from cells differentiated in culture for 4 d as in Figure 1. Note that activation (Mn+) increases the level of the α6β1 integrin in the DIG (raft) fraction but has no effect on integrin levels in the non-DIG compartment (left panel). Raft localization of α6β1 is also confirmed by the shift of the integrin into the non-DIG fraction after raft depletion using FB1 in the presence of Mn²⁺ (right panel). In contrast, immunoprecipitations using the F11 anti-αvβ3 Ab show that activation increases the level of αvβ3 in cells grown on Fn, but only in the non-DIG (nonraft) fraction (bottom panel). The arrow marks the position of the integrin band in each set of blots.

Figure 6.

The contributions of PI3K and MAPK pathways to oligodendrocyte survival signaling after raft depletion and integrin activation. Survival assays of oligodendrocytes on Ln-2 as in Figure 2 were used to measure the change in response to 1 ng/ml PDGF after raft depletion (FB1) and integrin activation (Mn) in association with pharmacological inhibition of PI3K with wortmannin (WM) or LY294002 (LY), or of MAPK with PD98059(PD). Data are presented as the mean percentage change relative to survival of control cells (cells grown on PDL in the absence of PDGF). For each experiment, cells were plated on PDL in the presence of PDGF and either left untreated (white bars) or treated with FB1, Mn²⁺, or a combination of the two (light gray, dark gray, and blackbars, respectively). The experiment is therefore designed to allow comparison of the relative contribution to PDGF-mediated survival of PI3K and MAPK signaling within a treatment group; the between-group comparison of survival is shown in Figure 2. Values are presented as mean ± SD of five independent experiments. Statistical significance was analyzed using the Student's t test. *p < 0.01, **p < 0.02, and ***p < 0.001 compared with control cells without inhibitors.