Perturbed interactions of mutant proteolipid protein/DM20 with cholesterol and lipid rafts in oligodendroglia: implications for dysmyelination in spastic paraplegia

Abstract

Missense mutations in the human PLP1 gene lead to dysmyelinating diseases with a broad range of clinical severity, ranging from severe Pelizaeus-Merzbacher disease (PMD) to milder spastic paraplegia type 2 (SPG-2). The molecular pathology has been generally attributed to endoplasmic reticulum (ER) retention of misfolded proteolipid protein (PLP) (and its splice isoform DM20) and induction of the unfolded protein response. As opposed to previous studies of heterologous expression systems, we have analyzed PLP/DM20 trafficking in oligodendroglial cells, thereby revealing differences between PMD and SPG-2-associated PLP/DM20 isoforms. PLP(A242V) and DM20(A242V) (jimpy-msd in mice), associated with severe PMD-like phenotype in vivo, were not only retained in the ER but also interfered with oligodendroglial process formation. In contrast, glial cells expressing SPG-2-associated PLP(I186T) or DM20(I186T) (rumpshaker in mice) developed processes, and mutant PLP/DM20 reached a late endosomal/lysosomal compartment. Unexpectedly, PLP/DM20 with either substitution exhibited impaired cholesterol binding, and the association with lipid raft microdomains was strongly reduced. Turnover analysis demonstrated that mutant PLP was rapidly degraded in oligodendroglial cells, with half-lives for PLP > PLP(I186T) > PLP(A242V). Protein degradation was specifically sensitive to proteasome inhibition, although PLP/DM20(I186T) degradation was also affected by inhibition of lysosomal enzymes. We conclude that, in addition to ER retention and unfolded protein response (UPR) induction, impaired cholesterol binding and lipid raft association are characteristic cellular defects of PLP1-missense mutations. Mutant protein is rapidly cleared and does not accumulate in oligodendroglial cells. Whereas UPR-induced cell death governs the PMD phenotype of the msd mutation, we propose that impaired cholesterol and lipid raft interaction of the rsh protein may contribute to the dysmyelination observed in SPG-2.

Figure 1.

Localization of PLP and DM20 in oligodendroglial and COS7 cells. Primary cultured oligodendrocytes (A), wild-type PLP and DM20 transfected Oli-neu cells (B, C), and wild-type PLP transfected COS7 cells (D) were costained with antibodies AA3 recognizing PLP (green) and LAMP1 (red; A–C) or calnexin (red; D). In A and C, confocal images are shown, and B is a deconvolved image. Insets show enlarged areas. In primary oligodendrocytes and in Oli-neu cells, PLP/DM20 is efficiently incorporated in the plasma membrane and localized in endosomes/lysosomes [plasma membrane and endosomes/lysosomes are primarily detected in distinct focal planes; plasma membrane localization of PLP/DM20 is shown in supplemental Fig. 2 (available at www.jneurosci.org as supplemental material)]. In COS7 cells, a large proportion of the protein is found in the ER. The dashed lines outline the position of the nuclei. The minor endosomal/lysosomal localization of PLP in COS7 is shown in supplemental Figure 2 (available at www.jneurosci.org as supplemental material). Scale bars, 5 μm. pOL, Primary oligodendrocyte; wt, wild type; Cnx, calnexin.

Figure 2.

Cell surface transport efficiency of wild-type and mutant PLP and DM20. PLP and DM20 transfected cells were stained live with the O10 antibody, which recognizes an extracellular epitope, and thus selectively marks cells, where PLP/DM20 is localized to the cell surface (right picture columns in A and C, “cell surface PLP/DM20”). After O10 surface staining, cells were fixed, permeabilized, and costained with the AA3 antibody, which binds to the cytoplasmic C terminus of PLP/DM20 and thus identifies all cells expressing PLP/DM20 (left picture columns in A and C, “total PLP/DM20”). Cells expressing PLP/DM20 that has not reached the plasma membrane are visualized in the staining with the AA3 antibody but do not stain with the O10 antibody. The O10-positive cells (“cell surface PLP/DM20”) were counted and expressed as a percentage of all PLP/DM20-expressing cells (“total PLP/DM20”), evaluating the surface transport efficiency of PLP/DM20 (B, D). A, B, Cell surface transport efficiency of wild-type PLP and DM20 expressed in COS7 cells and Oli-neu cells. In Oli-neu cells, wild-type PLP and DM20 are efficiently transported to the plasma membrane, whereas in a large fraction of COS7 cells PLP/DM20 is retained intracellularly. A total of 400–550 PLP/DM20-expressing cells was counted in each of three independent experiments. C, D, Cell surface expression of rsh- and msd-mutant PLP and DM20 in Oli-neu cells. A total of 350–600 PLP/DM20-expressing cells was counted in each of four independent experiments. Error bars represent SEM. Note that rsh-PLP and rsh-DM20 retain some capacity for cell surface transport (arrowheads). Scale bars, 30 μm.

Figure 3.

Distinct subcellular localization of rsh- and msd-PLP. Oli-neu cells were transfected with wild-type (wt), rsh-, and msd-PLP and colabeled using antibodies that recognize PLP (green) and the ER marker protein BiP (A; red) or LAMP1 (B; red). Deconvolved images are shown. Wild-type as well as rsh protein colocalize primarily with the lysosomal marker, whereas msd protein colocalizes with the ER marker. Insets show enlargements. Scale bars, 5 μm. Note dramatic differences in cellular phenotype induced by msd protein.

Figure 4.

Cholesterol interaction of mutant PLP/DM20. Oli-neu cells transfected with wild-type, rsh and msd mutant PLP (A), or DM20 (B) were stained with antibodies AA3 to PLP/DM20 (left pictures) and with the cholesterol interacting agent filipin, which is autofluorescent (right pictures). Expression of mutant PLP/DM20 does not induce redistribution of cholesterol. Scale bars, 10 μm. C, Transfected Oli-neu cells were labeled overnight with [³H]photocholesterol. Cells were UV-irradiated to covalently cross-link cholesterol binding proteins, and total cell lysates were directly analyzed by SDS-PAGE (top panel) or subjected to PLP/DM20 immunoprecipitation using AA3 antibodies (IP; bottom panel). [³H]Photocholesterol-modified proteins were visualized by autoradiography. The arrowheads mark the position of PLP and DM20 in the gel. D, [³H]Photocholesterol labeling and UV cross-linking of total membranes isolated from transfected Oli-neu cells. Proteins were separated by SDS-tube gel electrophoresis and slices were analyzed for associated radioactivity by scintillation counting. Results obtained from PLP/DM20 transfected (closed circles) are plotted over those from control cells (open circles). Peaks of radioactivity resulting from [³H]photocholesterol modification of transfected PLP or DM20 are marked by arrows. E, A Western blot of the same membrane fractions analyzed in Figure 4D with antibodies AA3 recognizing PLP/DM20 demonstrates that equal amounts of total PLP/DM20 were loaded.

Figure 5.

Impaired association of mutant PLP/DM20 with lipid rafts. Wild-type, rsh-, and msd-PLP (A) and DM20 (B) transfected Oli-neu cells were subjected to lipid raft analysis, by cell extraction in a CHAPS-containing buffer at 4°C followed by density gradient centrifugation. Fractions 1 (light fraction) to 6 (dense fraction) were collected and analyzed by Western blotting using antibodies recognizing PLP/DM20. Because of their low density, lipid rafts float in the light fractions of the gradient and are collected from fractions 1 and 2. A high proportion of wild-type PLP and DM20 is present in the lipid raft fraction, whereas rsh- and msd-PLP/DM20 are almost absent from this fraction. The asterisk (*) marks an endogenous DM20 band, which is occasionally seen in Western blots.

Figure 6.

Pulse-chase turnover analysis of wild-type and mutant PLP/DM20. Wild-type, rsh-, and msd-PLP or DM20 transfected Oli-neu cells were pulse labeled for 30 min with [³⁵S]methionine/cysteine followed by incubation in normal growth medium for indicated times (chase). Labeled PLP and DM20 were recovered by immunoprecipitation. A, Immunoprecipitates were analyzed by SDS-PAGE, and ³⁵S-labeled PLP/DM20 was detected by phosphoimaging. B, C, The recovery of ³⁵S-labeled PLP/DM20 was densitometrically quantified. The amount of PLP/DM20 recovered immediately after the pulse (0 h chase) was equalized to 100%, and the percentage of recovery after the different chase times was calculated. Error bars represent SEM. Statistical significance was determined using the t test (*p < 0.05; **p < 0.01; ***p < 0.001). rsh- and msd-PLP are significantly faster degraded than wild-type PLP, whereas degradation of mutant DM20 versus wild-type DM20 is not significantly enhanced (B). C illustrates the turnover of wild-type and mutant PLP compared with the respective DM20 isoform. n = 12 for PLP 4 h plus 8 h, n = 8 for DM20 4 h plus 8 h, n = 3 for PLP plus DM20 24 h.

Figure 7.

Proteasomal and lysosomal degradation of PLP and DM20. Wild-type and mutant PLP- or DM20-expressing Oli-neu cells were subjected to pulse-chase analysis as described in Figure 6. During the chase, the cells were either left untreated, or treated with the proteasome inhibiting peptide ALLN (100 μm), or the lysosomal enzyme inhibitor leupeptin (LEU) (100 μg/ml). A, Immunoprecipitation of wild-type and mutant PLP (left panels) and DM20 (right panels) analyzed by SDS-PAGE and phosphoimaging of ³⁵S-labeled PLP/DM20. B, Densitometric quantification of recovered PLP/DM20. Statistical significance was determined by applying the paired t test (*p < 0.05; **p < 0.01; ***p < 0.001). Wild-type PLP and DM20 are degraded by both the proteasome and the lysosome. Mutant PLP and DM20 are predominantly degraded by the proteasome. Significant lysosomal degradation of rsh-DM20 is observed. n = 8 for wild-type and rsh-PLP; n = 6 for msd-PLP; n = 3 for wild-type and rsh-DM20; n = 4 for msd-DM20. Error bars indicate SEM.

Figure 8.

Shift of the subcellular PLP localization by ALLN and leupeptin treatment. Wild-type, rsh- and msd-PLP transfected cells were treated with ALLN (100 μm) (A) or leupeptin (100 μg/ml) (B). After 8 h of treatment, cells were fixed and stained with antibodies to PLP/DM20 and calnexin (A) or LAMP1 (B). Note that ALLN or leupeptin treatment induces a shift in the subcellular localization of wild-type and rsh-PLP. Scale bars, 20 μm.