Dysfunction of Platelet-derived Growth Factor Receptor α (PDGFRα) Represses the Production of Oligodendrocytes from Arylsulfatase A-deficient Multipotential Neural Precursor Cells*

Background: PDGFRα is a key signaling component in oligodendrogenesis. Results: Multipotential Neural precursors (NPs) deficient in arylsulfatase A (ASA) show a higher ratio of long versus short fatty acid sulfatides, reduction in PDGFRα, decreased AKT phosphorylation, and increased exosomal shedding of PDGFRα. Conclusion: Sulfatides are regulators of NP response to PDGF-AA. Significance: A novel regulatory mechanism of PDGFRα signaling is described. The membrane-bound receptor for platelet-derived growth factor A (PDGFRα) is crucial for controlling the production of oligodendrocytes (OLs) for myelination, but regulation of its activity during OL differentiation is largely unknown. We have examined the effect of increased sulfated content of galactosylceramides (sulfatides) on the regulation of PDGFRα in multipotential neural precursors (NPs) that are deficient in arylsulfatase A (ASA) activity. This enzyme is responsible for the lysosomal hydrolysis of sulfatides. We show that sulfatide accumulation significantly impacts the formation of OLs via deregulation of PDGFRα function. PDGFRα is less associated with detergent-resistant membranes in ASA-deficient cells and showed a significant decrease in AKT phosphorylation. Rescue experiments with ASA showed a normalization of the ratio of long versus short sulfatides, restored PDGFRα levels, corrected its localization to detergent-resistant membranes, increased AKT phosphorylation, and normalized the production of OLs in ASA-deficient NPs. Moreover, our studies identified a novel mechanism that regulates the secretion of PDGFRα in NPs, in glial cells, and in the brain cortex via exosomal shedding. Our study provides a first step in understanding the role of sulfatides in regulating PDGFRα levels in OLs and its impact in myelination.

The membrane-bound receptor for platelet-derived growth factor A (PDGFR␣) is crucial for controlling the production of oligodendrocytes (OLs) for myelination, but regulation of its activity during OL differentiation is largely unknown. We have examined the effect of increased sulfated content of galactosylceramides (sulfatides) on the regulation of PDGFR␣ in multipotential neural precursors (NPs) that are deficient in arylsulfatase A (ASA) activity. This enzyme is responsible for the lysosomal hydrolysis of sulfatides. We show that sulfatide accumulation significantly impacts the formation of OLs via deregulation of PDGFR␣ function. PDGFR␣ is less associated with detergentresistant membranes in ASA-deficient cells and showed a significant decrease in AKT phosphorylation. Rescue experiments with ASA showed a normalization of the ratio of long versus short sulfatides, restored PDGFR␣ levels, corrected its localization to detergent-resistant membranes, increased AKT phosphorylation, and normalized the production of OLs in ASA-deficient NPs. Moreover, our studies identified a novel mechanism that regulates the secretion of PDGFR␣ in NPs, in glial cells, and in the brain cortex via exosomal shedding. Our study provides a first step in understanding the role of sulfatides in regulating PDGFR␣ levels in OLs and its impact in myelination.
Oligodendrocyte progenitor cells (OPCs) 2 are primarily generated from undifferentiated multipotential neural precursors (NPs) during early embryogenesis under the influence of morphogenic molecules, such as Shh (sonic hedgehog) (1)(2)(3)(4). Multiple mechanisms are developmentally coordinated to regulate the differentiation of OPCs into functional oligodendrocytes (OLs) (5)(6)(7). Of these, signaling through the platelet-derived growth factor receptor ␣ (PDGFR␣) is essential in controlling the proliferation and survival of OPCs (8 -10). PDGFR␣ is expressed in NPs (11) and marks the appearance of the first PDGF-responsive OPCs during early neurogliogenesis (12). Numerous studies have highlighted the relevance of PDGFR␣ signaling in oligodendrogenesis. For example, PDGFR␣ ϩ cells isolated from the embryonic spinal cord exclusively generate myelinating oligodendrocytes (12,13). Furthermore, the formation of mature OLs is significantly reduced upon ablation of PDGFR␣ ϩ cells (12) and in mice with targeted disruption of its ligand, PDGF-AA (14). Functionally, the activity of PDGFR␣ depends in part on its localization to detergent-resistant membrane (DRM) domains in the plasma membrane and interaction with integrin complexes (15)(16)(17). The association of PDGFR␣ with DRMs underlines the possibility that conditions affecting DRMs may impact PDGFR␣ function and, therefore, the myelination program.
Previously, we found that exogenous sulfated galactosylceramides (sulfatides) significantly reduced the formation of OLs from NPs in vitro (18). One interpretation from these results is that sulfatides might repress the sensitivity of OPCs to proliferating signals such as PDGF-AA, thereby reducing the pool of progenitors available for differentiation into mature OLs. Sulfatides are crucial sphingolipids in myelin architecture (19) and have been found to negatively regulate the maturation of OPCs into differentiated OLs (20,21), but the mechanism is still unknown. Sphingolipids, including sulfatides, have structural and functional roles in DRMs (22,23) and participate in caveolar and exosomal biogenesis (22, 24 -26). Therefore, sulfatides have an intrinsic potential to modulate the activity of membrane-bound receptors such as PDGFR␣ by altering membrane domains such as DRMs.
In this study, we tested the hypothesis that sulfatides contribute to the regulation of oligodendrogenesis by modulating PDGFR␣ function. We found that increased sulfatide levels in NPs lead to a reduced production of OPCs and OLs. Furthermore, we observed a diminished association of PDGFR␣ with DRMs, repressed AKT phosphorylation, and exacerbated secretion of PDGFR␣ via exosomes. We present evidence that exosomal secretion of PDGFR␣ is a natural process in glial cells in vitro and during myelination of the murine cortex, when sulfatides are highly produced.

EXPERIMENTAL PROCEDURES
Animals-Heterozygous ASA ϩ/Ϫ breeders (obtained from Dr. Gieselmann and back-crossed in the C56BL/6 background) were maintained in standard housing conditions, under the approval of the Animal Care and Use Committee. ASA ϩ/ϩ and ASA Ϫ/Ϫ embryos at 16.5 days of gestation and 3-day-old newborns were used in our experiments. ASA ϩ/ϩ and ASA Ϫ/Ϫ mice at 7, 14, and 21 days were killed without sex distinctions for immunocytochemical and in vivo exosome isolation studies.
Multipotential Neural Precursor Preparations and Cell Culture Conditions-NPs were isolated from ASA ϩ/ϩ and ASA Ϫ/Ϫ embryonic day 16.5 telencephalon by mechanical dissociation and maintained as proliferating spheres in the presence of 10 ng/ml FGF-2 and 20 ng/ml EGF (27). Cultures of NPs obtained from different litters were used between passages 3 and 10 with identical results (n ϭ 5-6). For differentiation assays, NPs were mechanically dissociated and seeded at a density of 7.5 ϫ 10 4 cells/cm 2 onto coverslips precoated with Matrigel (BD Biosciences) for 1 h at room temperature. Cultures were maintained for 3 or 7 days (3 or 7 DIV) in the absence of growth factors and in the presence of 2% fetal bovine serum (differentiated medium). Differentiated medium containing 2% FBS showed traces of Alix and Rab5B (data not shown). In some experiments, differentiated cells were exposed to PDGF-AA (Peprotech) at a concentration of 20 ng/ml for 1 day after plating. For Western blot analyses, NP spheres were collected 5 days after proliferation or 7 days after plating for differentiation.
Studies of PDGFR␣ Proteolysis-Analysis of proteolytic degradation of the PDGFR␣ were performed utilizing 2 ϫ 10 6 dissociated ASA ϩ/ϩ and ASA Ϫ/Ϫ NPs. NPs were exposed to 10 M MG132 or 10 mM NH4CL in basal proliferating medium conditions. Cells incubated with MG132 or NH4Cl for 6 h were collected for protein expression analysis of the PDGFR␣ as described below (see "Immunoblotting"). Because MG132 was dissolved in DMSO, DMSO-treated ASA ϩ/ϩ and ASA Ϫ/Ϫ NPs were included as controls. All experiments were repeated three times. Additionally, three independent experiments were performed exposing 4 ϫ 10 6 dissociated ASA ϩ/ϩ and ASA Ϫ/Ϫ NPs to PDGF-AA ligand at a concentration of 20 ng/ml for 30 min before cell collection. For these experiments, NPs were starved for 3 h of growth factors present in the proliferating medium (EGF and basic FGF) and exposed to MG132 or NH 4 Cl as described above. After starvation, NP metabolism was slowed down by an ice bath for 15 min, cells were pelleted, and medium was preserved on ice. Pelleted NPs were exposed to fresh medium containing PDGF-AA for 30 min on ice. Cells were pelleted and washed to remove unbound ligand and resuspended in their original medium with MG132 or NH 4 Cl for an additional 30 min at 37°C. NPs were pelleted, washed with PBS, and collected for Western blot analysis. NP pellets were analyzed for PDGFR␣ and downstream signaling for AKT, phospho-AKT (Ser 308 and Thr 478 ), MAPK p42/44, and phospho-MAPK p42/44 (Thr 202 /Tyr 204 ).
Sulfatide Treatment-For analysis of the effects of exogenous sulfatides (Avanti), ASA ϩ/ϩ NP spheres were dissociated and incubated with two pulses of 10 M sulfatides at days 1 and 3 and collected after 5 days.
Mouse Glial Primary Culture-Primary glial cultures were prepared from ASA ϩ/ϩ and ASA Ϫ/Ϫ as described previously (28). Cell suspensions were seeded in 75-cm 2 tissue culture flasks coated with 10 g/ml poly-L-lysine at equal cell densities. Medium was changed 4 days after plating. Glial conditioned medium was collected at 7, 14, and 21 DIV and stored at Ϫ20°C.
CG4 Cell Culture-CG4 cells were cultured as described previously (29), except that B104 conditioned medium was replaced with 10 ng/ml PDGFA (BD Biosciences) and 5 ng/ml basic FGF (Peprotech). After 5 days in culture, supernatants were processed for exosomal isolation and cell pellets for Western blot analysis as described below.
Detergent-resistant Membranes Isolation-NP lysates (equivalent to 2 mg of protein) were equilibrated with a solution of 90% sucrose to give a final solution of 45% sucrose and subjected to ultracentrifugation using a sucrose gradient (24). Twelve 0.25-ml fractions were collected and analyzed by immunoblotting.
Cholesterol Assay-Cholesterol levels of ASA ϩ/ϩ and ASA Ϫ/Ϫ NPs from each sucrose fraction (50 l) were measured using the fluorometric Amplex Red cholesterol assay kit (Invitrogen). Fluorometric analysis was performed using the Beckman Coulter DTX 880 multimode detector.
Exosome Preparation and Transmission Electron Microscopy-Conditioned media from NPs (3 ϫ 10 7 cells) and primary glial cultures (3 ϫ 10 7 ) (n ϭ 3 for each group) were filtered (0.22 m) and subjected to ultracentrifugation at 100,000 ϫ g for 90 min. Pellets were resuspended in cold PBS and ultracentrifuged at 100,000 ϫ g for 90 min. Exosome pellets were dissolved in lysis buffer as described below, and equal amounts of proteins (4 g) were separated by SDS-PAGE and transferred to nitrocellulose, followed by immunodetection of PDGFR␣, EGFR, and the exosomal marker Rab-5b. Exosomes were isolated from ASA ϩ/ϩ and ASA Ϫ/Ϫ brain cortices at 7, 14, and 21 days postnatal as described (34). The final exosomal pellet was subjected to a sucrose gradient centrifugation at 200,000 ϫ g for 16 h at 4°C. A total of seven fractions were collected (fraction a, top; fraction g, bottom). Fractions b, c, and d, enriched in the exosomal marker Rab-5b, were each resuspended in 40 l of lysis buffer. Western blot analysis was performed on a half of the volume of each fraction. For transmission electron microscopy, exosomes were fixed in 2% paraformaldehyde and stained as described (35). Samples were imaged using a 120-kV transmission electron microscope, JEOL JEM-1220, and equipped with a Gatan Es1000W 11-megapixel CCD camera.
Rescue of ASA Deficiency in ASA Ϫ/Ϫ NPs-The deficiency in ASA activity in ASA Ϫ/Ϫ NPs was rescued by cross-correction using ASA-supplemented cell medium (36). Briefly, ASA-secreting HeLa cells were grown in 10% FCS DMEM/F-12 before conditioning. Cells were incubated with serum-free cell medium for 4 days. Conditioned media were filtered through a 0.45-m filter and kept frozen until use. For correction experiments, 5, 15, or 25% ASACM or 25% control medium (DMEM/ F-12 medium) was added to ASA Ϫ/Ϫ NP culture medium. ASA activity was measured utilizing 4-methylumberyl sulfate derivative substrate as described by Porter et al. (37).
Immunoblotting-Samples were homogenized in lysis buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton, mammalian protease inhibitor mixture, 1 mM PMSF, 1 mM okadaic acid, and 2 mM sodium orthovanadate) on ice for 30 min with vortexing at 10-min intervals. After centrifugation at 10,000 ϫ g, 10 g of protein/sample was loaded onto 4 -12% 1.5-mm precast NuPage gels (Invitrogen) and electrophoresed using an XCell Sure-lock vertical electrophoresis system (Invitrogen). After transferring to nitrocellulose membrane, blots were blocked with milk/BSA solution and incubated with the relevant antibodies. Immunoreactive products were detected using peroxidase-labeled secondary antibodies and ECL chemiluminescent substrate (Pierce). Immunoblots were semiquantified using ImageJ analysis software (National Institutes of Health).
Immunocytochemistry and Immunohistochemistry-For differentiation studies, cultures were maintained for 3 or 7 DIV in the absence of growth factors and in the presence of 2% fetal bovine serum. Cells were fixed with 4% paraformaldehyde for 20 min and washed twice with PBS. Cells were incubated with primary antibodies at room temperature for 1 h or overnight at 4°C, washed extensively with PBS, and then incubated with Alexa-conjugated secondary antibodies (Molecular Probes). Coverslips or chamber slides were mounted with Prolong Gold antifade reagent with DAPI (Invitrogen) and imaged using either DM5500 Q Microscope with a Leica DFC500 camera or Zeiss Meta 510 confocal microscope. Cell counting was performed in the ImageJ software by counting cell-specific markers (NG2 for oligodendrocyte precursor cells, O4 for intermediate oligodendrocytes, glial fibrillary acidic protein (GFAP) for astrocytes, and DAPI for the nuclei). Three images of each coverslip or chamber slide were counted, and all experiments were repeated at least three times with different primary NPs cultures. The cell percentage was calculated for each specific marker with respect to total DAPI ϩ cells. Brains were collected after perfusion with PBS and 4% paraformaldehyde. Sections were cryoprotected in 20% sucrose, embedded in OCT, and sectioned at 20 m on a cryostat. Tissue sections were immunostained for myelin basic protein (MBP) and PDGFR␣ and developed using the diaminobenzidine-ABC kit (Vector Laboratories).
Antibodies-The following antibodies were used in this study: actin (1:3000; Sigma), AKT (1:1000; Cell Signaling), Alix Statistical Analysis-Data were analyzed by Student's t test or one-way analysis of variance, considering a p value of Ͻ0.05 as significant. Results are given as the means Ϯ S.E. All analyses were performed using GraphPad Prism version 5.0d (GraphPad Software, Inc., La Jolla, CA).

Levels of PDGFR␣ and Myelin Proteins Are Reduced during
Active Myelination in ASA Ϫ/Ϫ Animals-Previous studies performed in ASA Ϫ/Ϫ animals during the peak of myelination showed a delayed expression of mRNA for myelin proteins: MBP, myelin and lymphocyte protein, and PLP, which suggests a defect in the differentiation program of OLs in the ASA Ϫ/Ϫ context (38). We have examined the expression of PDGFR␣ and MBP by immunohistochemistry in the cortex of ASA ϩ/ϩ and ASA Ϫ/Ϫ mice during the period of active myelination at postnatal days 7, 14, and 21 ( Fig. 1, A and B). Our analysis showed a reduced content of the PDGFR␣ (Fig. 1A) and MBP (Fig. 1B) in the ASA Ϫ/Ϫ cortex. Immunoblotting analysis confirmed the immunohistochemical findings. PDGFR␣ protein levels were significantly reduced in the mutant cortex at all ages examined (Fig. 1, C and F). MBP (Fig. 1, D and F) and PLP (Fig. 1E) were also significantly reduced in the ASA Ϫ/Ϫ brain.
ASA Deficiency Alters Gliogenesis in Vitro-To better understand the possible effect of ASA deficiency on the production of OLs, multipotential NPs were isolated from embryonic day 16.5 of ASA Ϫ/Ϫ and ASA ϩ/ϩ mice, and NPs were differentiated for 7 days. ASA Ϫ/Ϫ NPs produced significantly fewer O4 ϩ intermediate OLs (Fig. 2, A and B) and more GFAP ϩ astrocytes (Fig. 2, A and B) than cells with normal levels of ASA activity. A marked decrease of immature NG2 ϩ OPCs was also determined in ASA Ϫ/Ϫ cells differentiated for 3 DIV (3.4 Ϯ 0.6% in ASA Ϫ/Ϫ cells versus 8.3 Ϯ 0.5% in ASA ϩ/ϩ cells), indicating that the decrease in the production of oligodendroglial cells starts at early stages of in vitro differentiation. Sulfatide-treated ASA ϩ/ϩ NPs showed a significant reduction in the numbers of O4 ϩ cells as was observed in differentiated ASA Ϫ/Ϫ NPs (Fig.  2B). Immunoblotting analysis of protein extracts from differentiated ASA Ϫ/Ϫ NPs confirmed a failure of these cultures to produce OLs that express MBP (Fig. 2C). Measurement of four major sulfatide isoforms by UHPLC-MS/MS showed a 2-fold significant increase in sulfatides in ASA Ϫ/Ϫ NP spheres with respect to ASA ϩ/ϩ NP spheres (Fig. 2D).
The PDGFR␣ Pathway Is Defective in ASA Ϫ/Ϫ Cells-Because the PDGFR␣ is a critical signal for the generation of OPCs and their timely maturation into OLs, we studied its expression in ASA Ϫ/Ϫ NPs. PDGFR␣ is crucial for regulating the number and survival of OPCs/OL during myelination (9 -11, 39 -41), mainly by regulating the activity of the AKT or ERK1/2 pathway (42)(43)(44). To determine whether this receptor was involved in the phenotype of ASA Ϫ/Ϫ cells, various experiments were performed. Immunoblotting analyses showed a significant reduction of PDGFR␣ protein levels in proliferating and differentiating ASA Ϫ/Ϫ cells (Fig. 3A). This decrease appeared specific to PDGFR␣ because control experiments did not show significant changes in levels of EGFR (Fig. 3A). Next, we measured the response of ASA Ϫ/Ϫ cells to the mitogenic stimulus of exogenous PDGF-AA. Differentiating ASA Ϫ/Ϫ and ASA ϩ/ϩ NP cultures were incubated in the presence of PDGF-AA and pulsed with BrdU on day 2. Immunocytochemistry of NG2 showed significantly fewer BrdU ϩ /NG2 ϩ OPCs in ASA Ϫ/Ϫ cultures (Fig. 3B). Immunoblotting analysis of PDGFR␣ levels showed reduced levels of the receptor in PDGF-AA-stimulated NPs (Fig. 3C), confirming a failure of ASA Ϫ/Ϫ cells to respond to the growth factor. Functional studies of PDGFR␣ looking at AKT and MAPK ERK1/2 phosphorylation (Fig. 3, D-F) confirmed a severe failure of ASA Ϫ/Ϫ cells to phosphorylate serine 473 and threonine 308 residues of AKT upon stimulation with PDGF-AA (Fig. 3E).
The Transcription and Degradation of the PDGFR␣ Are Not Affected in ASA Ϫ/Ϫ Cells-Our previous experiments indicated a reduction of PDGFR␣ in ASA Ϫ/Ϫ -deficient NPs. This reduction could be caused by different defects affecting protein production (e.g. gene transcription), degradation (e.g. proteosome), and secretion (45)(46)(47). Real-time PCR analyses of PDGFR␣ mRNA levels (Fig. 4A) and immunoblotting analyses for stress response markers (heat shock proteins HSP40 and HSP60; not shown) did not find significant differences in ASA Ϫ/Ϫ and ASA ϩ/ϩ NPs. Furthermore, blockage of proteasome activity with MG132 and of lysosomal function with NH4Cl showed no significant differences in PDGFR␣ protein levels in ASA Ϫ/Ϫ cells grown in basal culture conditions or stimulated with PDGF-AA (Fig. 4, B and C). Together, these results indicate that defects in gene transcription, stress-related gene translation deficiencies, and altered protein degradation are unlikely to be causes for the reduction of PDGFR␣ protein levels in ASA Ϫ/Ϫ cells. PDGFR␣ Is Secreted via Exosomal Shedding-Receptor shedding via exosomal secretion has been shown to be an important mechanism by which cells regulate signaling with their environment (48). Exosomes are vesicles of ϳ20 -100 nm in diameter generated through the formation of multivesicular bodies (49). Of relevance, OPCs and OLs are known to shed various proteins and lipids through exosomal secretion in vitro (50,51). To determine whether PDGFR␣ is associated with this process, exosomes were prepared from conditioned cell culture media of ASA ϩ/ϩ and ASA Ϫ/Ϫ NPs. Electron microscopy examination of exosomal preparations showed that ASA Ϫ/Ϫ and ASA ϩ/ϩ have similar stereotypical profiles of circular vesicles with diameters ranging from ϳ25 to 100 nm (Fig. 4, D and E). Immunoblotting analysis of exosomal protein extracts showed significant increase in PDGFR␣ levels in ASA Ϫ/Ϫ exosomes (Fig. 4F), which represents about 3-4% of the reduction in PDGFR␣ observed in ASA Ϫ/Ϫ NPs. In contrast, receptor levels in ASA ϩ/ϩ exosomes were significantly lower in ASA ϩ/ϩ and FIGURE 3. Loss of PDGF-AA response in NPs with high sulfatide content. A, immunoblotting analyses for the expression of PDGFR␣, EGFR, and actin in ASA ϩ/ϩ and ASA Ϫ/Ϫ NPs. Analysis was performed in proliferating NPs after 5 DIV and in differentiated cells after 7 DIV in the presence or absence of 20 ng/ml PDGF-AA. PDGFR␣ levels were reduced in all ASA Ϫ/Ϫ NP conditions. B, ASA ϩ/ϩ and ASA Ϫ/Ϫ NP spheres were dissociated for differentiation and incubated with or without 20 ng/ml PDGF-AA and pulsed with BrdU. Proliferating oligodendrocyte progenitors were identified by co-localization of NG2 ϩ and BrdU ϩ at 3 days using immunocytochemistry. BrdU ϩ /NG2 ϩ cells were significantly reduced in ASA Ϫ/Ϫ cultures (data shown as the mean Ϯ S.E. (error bars); n ϭ 3). *, p Ͻ 0.05 by Student's t test). C, semiquantification of immunodetected PDGFR␣ in differentiated ASA Ϫ/Ϫ cells. In contrast to control cells, incubation with PDGF-AA did not increase receptor levels (data shown as the mean Ϯ S.E.; n ϭ 3). ASA ϩ/ϩ versus ASA Ϫ/Ϫ (Ϫ) PDGFA, *, p ϭ 0.038; ASA ϩ/ϩ versus ASA Ϫ/Ϫ (ϩ) PDGF-AA, *, p ϭ 0.0025 by Student's t test. D-F, ASA ϩ/ϩ and ASA Ϫ/Ϫ NPs were starved of growth factors for 3 h and exposed to PDGF-AA ligand for 30 min at 4°C. Unbound ligand was washed off, and cells were incubated for 30 min at 37°C. AKT phosphorylation at Thr 308 and Ser 473 was significantly decreased in ASA Ϫ/Ϫ NPs with respect to ASA ϩ/ϩ NPs. ERK1 phosphorylation at Thr 202 and ERK2 Tyr 204 was similar in both cell types (data shown as the mean Ϯ S.E.; n ϭ 3). *, p Յ 0.05; **, p Յ 0.01 by Student's t test. absent in mock medium (Fig. 4, F-H). Treatment of ASA ϩ/ϩ NPs with 10 M exogenous sulfatides also led to an exosomal release of PDGFR␣, although less intense than in ASA Ϫ/Ϫ cells, possibly due to ASA activity in ASA ϩ/ϩ cells (Fig. 4F). Levels of EGFR in exosomes were significantly reduced in ASA Ϫ/Ϫ when normalized against the exosomal housekeeping protein Alix but not against Rab-5b (Fig. 4, G and H). This may reflect compositional changes in exosomes from mutant cells.
Reduced PDGFR␣ in Detergent-resistant Membranes of ASA Ϫ/Ϫ Cells-Association of PDGFR␣ with DRMs is needed for its function in OPCs (33,34). To examine whether this association was affected in ASA Ϫ/Ϫ cells, a Lubrol-sucrose gradient method that fractionates plasmalemma DRMs from other non-DRM membranes (lysosomes, Golgi apparatus, etc.) was used (52). Immunoblotting analyses showed a sharp reduction (ϳ5% of the total level of receptor) of PDGFR␣ in DRMs (fractions 4 -6) from ASA Ϫ/Ϫ cells (Fig. 5A). DRM markers flotillin 2 and caveolin 1 were also significantly reduced in mutant DRMs (Fig.  5B). Interestingly, exposure of ASA ϩ/ϩ cells to sulfatides led to reductions of PDGFR␣ in DRMs (Fig. 5A). The total level of cholesterol (another DRM compound) and its buoyancy were not significantly different between ASA Ϫ/Ϫ and ASA ϩ/ϩ (Fig.  5C). Sulfatides were significantly increased in DRM fractions but decreased in non-DRM fractions from ASA Ϫ/Ϫ cells (Fig.  5D). Together, these results suggest that changes in DRM composition occur in ASA Ϫ/Ϫ cells, contributing to a reduction of PDGFR␣ in the plasma membrane.
Enzyme Correction Ameliorates Gliogenic and PDGFR␣ Defects in ASA Ϫ/Ϫ Cells-To evaluate whether gliogenic and receptor deficits can be corrected in ASA Ϫ/Ϫ cells, enzyme replacement was done by treating ASA Ϫ/Ϫ cells in culture medium conditioned from ASA prepared from ASA-overex-  t test). B and C, the effect of proteolytic inhibitor MG132 and NH4CL lysosomal inhibitor on the expression of PDFGR␣ showed similar recovery of receptor levels in ASA ϩ/ϩ and ASA Ϫ/Ϫ NPs in basal conditions and following PDGF-AA-stimulated conditions (data shown as the mean Ϯ S.E.; n ϭ 3). D and E, exosomes isolated from ASA ϩ/ϩ (D) and ASA Ϫ/Ϫ NP (E) culture media showed a typical vesicular shape with diameters between 30 and 100 nm. Bars, 50 nm. F, immunoblotting analysis showed the presence of higher levels of PDGFR␣ in exosomes released from ASA Ϫ/Ϫ NPs and from ASA ϩ/ϩ NP spheres treated with 10 M sulfatides. G and H, the relative abundance of PDGFR␣ and of EGFR were calculated with respect to the exosomal content of Rab-5b and Alix (data shown as the mean Ϯ S.E.; n ϭ 3). *, p Յ 0.05 by Student's t test.
pressing cells (ASACM) (36). This cross-correction experiment takes advantage of the general property of lysosomal enzymes to be taken up by cells, primarily via the endocytic mechanism involving a mannose 6-phosphate receptor (53). Cross-correction using this approach has been well established with a significant number of lysosomal enzymes, including ASA (54). ASA Ϫ/Ϫ NP spheres were incubated with 5, 10, or 25% ASACM before analysis. Immunocytochemical detection of ASA protein by confocal microscopy showed intracellular presence of ASA in corrected ASA Ϫ/Ϫ cells (Fig. 6A). ASA enzyme activity was substantially recovered (data not shown). Although sulfatide content was not normalized, there was a significant recovery of the normal ratio of long versus short fatty acid sulfatides in cells cultured with 25% ASACM (Fig. 6B).
Levels of PDGFR␣ were significantly normalized in corrected ASA Ϫ/Ϫ cells (Fig. 6C). Recovery of PDGFR␣ led to significant relocalization of the receptor in DRM fractions and less exosomal shedding (Fig. 6D). A significant recovery of AKT phosphorylation at serine residue 473 was observed but not in ERK1/2 phosphorylation (Fig. 6, E and F). Upon differentiation, production of O4 ϩ OLs from corrected ASA Ϫ/Ϫ cells was significantly normalized (Fig. 6G).
Exosomal Secretion of PDGFR␣ Occurs in Oligodendroglial Cells-Our experiments of exosomal shedding are relevant because this may be a regulatory mechanism used by OLs to locally and rapidly control levels of receptor available at the cell surface. To study this process in more detail, we measured receptor levels in exosomes isolated from primary glial cultures, CG4 cells (central glial-4 cells) and in the murine cortex. First, mixed glial cultures were prepared from newborn ASA ϩ/ϩ and ASA Ϫ/Ϫ pups and maintained for 7, 14, and 21 DIV, to enrich immature A2B5 ϩ /NG2 ϩ OPCs (7 DIV) or intermediate O4 ϩ , O1 ϩ , and mature MBP ϩ OLs (14 and 21 DIV), respectively (28,55). Quality control of OL differentiation in these conditions was confirmed by Western blot analysis of MBP expression (Fig. 7A). Whereas ASA ϩ/ϩ glial cultures showed the expected increase of MBP during culture differentiation, OL differentiation was markedly reduced in ASA Ϫ/Ϫ glial cultures (Fig. 7A). Exosomes were isolated from conditioned medium at each time point and analyzed for PDGFR␣ content by immunoblotting. PDGFR␣ was found in low but significant levels in exosomes from ASA ϩ/ϩ glia (Fig. 7, B and C). As expected, PDGFR␣ was increased in exosomes from ASA Ϫ/Ϫ glia (Fig. 7, B and C). A similar analysis was done with conditioned medium from cultures of oligodendroglial CG4 cells. Cells were grown in proliferating conditions for 5 DIV before analysis of exosomes. Immunodetectable levels of PDGFR␣ were present in the conditioned medium (Fig. 7D), indicating that this process occurs in OPCs independently of astrocytes being present.
Finally, we examined the extent to which exosomal shedding of PDGFR␣ occurs during myelination in the mouse brain. For this, we adapted a previously described technique (34). Transmission electron microscopy imaging confirmed the exosomal ultrastructure of these preparations from ASA ϩ/ϩ and ASA Ϫ/Ϫ brain material (Fig. 7E). Subsequent immunoblotting analysis showed the presence of PDGFR␣ (Fig. 7F) in brain exosomes from both genotypes. Exosomal shedding of PLP was used as a quality control for our experiments (Fig. 7G). This analysis showed a peak of the receptor in exosomes isolated from the cortex of P14 ASA ϩ/ϩ brain (Fig. 7F), a trend that was also observed in primary glial cell cultures (Fig. 7C). In contrast, PDGFR␣ levels were higher in exosomes from the ASA Ϫ/Ϫ cor- tex. Altogether, these results provide evidence that PDGFR␣ is exosomally secreted in vivo during the peak of myelination. Contextual changes, such as sulfatide accumulation, that alter this process may be relevant to understand possible defects in myelination in the brains of metachromatic leukodystrophy patients and in other demyelinating conditions.

DISCUSSION
This study provides insight into the interaction between sulfatides and PDGFR␣ activity in NPs. Experimental conditions that genetically or exogenously lead to increased sulfatide content revealed 1) defects in the association of PDGFR␣ with DRMs, 2) impaired translation of PDGF-AA signaling via repression of PDGFR␣/AKT activation, 3) increased shedding of PDGFR␣ via exosomes, and 4) decreased formation of NG2 ϩ OPCs and O4 ϩ OLs.
Various studies have shown that OL differentiation could either be promoted by a deficient synthesis of sulfatides (56,57) or inhibited by anti-sulfatide antibodies (58,59), suggesting that sulfatides negatively regulate OL differentiation (20,21). However, the mechanism of this repression has remained unaddressed. Our results using ASA Ϫ/Ϫ NPs with intrinsically high levels of sulfatides support the idea that these lipids are associated with reduced capacity to generate OLs, in line with previous in vivo analyses (60).
Although sulfatides are major components of mature myelin, they are synthesized during neural development in two distinct waves, first of short and then long fatty acid sulfatides, coinciding with the appearance of immature OPCs and more differentiated OLs, respectively (61). The physiological relevance of this change remains unclear. Recently published studies from our laboratory showed that short (C16:0 and C18:0) and long FIGURE 6. ASA-corrected ASA ؊/؊ NPs and recovery of PDGFR␣. A, confocal immunocytochemical analysis of ASA (in green) showed its association with Lamp1 ϩ lysosomes (in red) in ASA ϩ/ϩ NPs and the absence of ASA expression in ASAϪ/Ϫ NPs. ASAϪ/Ϫ cells treated with 25% ASA conditioned medium exhibited association with lysosomes (yellow). B, ASA-corrected ASAϪ/Ϫ NPs showed a significant reduction in the ratio of long (C24:0 and C24:1) versus short (C16:0 and C18:0) fatty acid isoforms in treatments with 25% conditioned medium (ASACM) but not with 5 or 15% ASACM (n ϭ 3). *, p Յ 0.05; **, p Յ 0.01 by analysis of variance. C, immunoblotting analysis of ASA-corrected ASA Ϫ/Ϫ NPs showed a normalization of PDGFR␣ levels in NPs cell lysates. D, PDGFR␣ protein expression levels also recovered in DRMs and decreased in exosomes from enzymatically corrected ASA Ϫ/Ϫ NPs. E, AKT Ser 473 but not Thr 308 showed a significant decrease in phosphorylation in ASA Ϫ/Ϫ NPs. F, ERK1 phosphorylation at Thr 202 , ERK2 at Tyr 204 was similar in both cell types (data shown as the mean Ϯ S.E.; n ϭ 3). *, p Ͻ 0.05 by Student's t test. G, enzymatically corrected ASA Ϫ/Ϫ NPs generated significantly more O4 ϩ OLs than untreated ASA Ϫ/Ϫ NPs 7 days after differentiation (data shown as the mean Ϯ S.E.; n ϭ 3). *, p Յ 0.05 by Student's t test.
(C24:0 and C24:1) fatty acid sulfatides are components of DRMs isolated from ASA ϩ/ϩ and ASA Ϫ/Ϫ brains and that their relative abundance is developmentally regulated (32). We propose that variations in short/long sulfatide isoforms may affect the fluidity of DRMs and induce changes in PDGFR␣ signaling (60,62,63). Our findings of a significant reduction of the ratio of long versus short fatty acid sulfatides associated with a normalization of the PDGFR␣ levels and AKT phosphorylation in ASA-corrected NPs support this hypothesis.
PDGFR␣ regulates the proliferation, survival, and differentiation of OPCs and OLs (10,11,39,64,65), a function that is dependent on its association with DRMs (16). Interestingly, PDGFR␣ membrane localization responds to changes in sphingolipid content (66). For example, ganglioside GM1 induced the relocation of PDGFR␣ to non-DRM/caveolar membranes, inhibiting PDGFR␣ signaling (67). Our study identified sulfatides as another group of sphingolipids with the capacity to reduce the association of the PDGFR␣ with DRMs and, consequently, impair the sensitivity of ASA Ϫ/Ϫ NPs to PDGF-AA. This defect did not appear to be caused by a reduction in gene transcription, increased degradation, or activation of cellular stress responses to the accumulation of sulfatides but rather to the impaired activity of the PDGFR␣/AKT pathway. This most likely stems from lowered association of the receptor with DRMs in ASA Ϫ/Ϫ NPs. Interestingly, the survival/expansion of OPC mediated by PDGF-AA involves the activation of AKT through phosphorylation of residues Thr 308 and Ser 473 (68,69). Our results demonstrate that in the presence of high levels of sulfatides, the relocalization of PDGFR␣ to non-DRM domains and phosphorylation of Thr 308 and Ser 473 of AKT are severely compromised. These findings further underline the importance of DRM integrity in AKT signaling activation (70,71). Although the molecular mechanism by which sulfatides reduce the association of the receptor in DRMs is unclear, the involve- Immunoblotting analysis showed the presence of PDGFR␣ in exosomes from cells with normal or increased sulfatide content. Rab-5b was used as an exosomal marker. C, exosomal secretion of PDGFR␣ is a naturally occurring event in cells with normal sulfatide content (ASA ϩ/ϩ ). Release of the receptor continuously increased as ASA Ϫ/Ϫ glial cultures matured in vitro (data shown as the mean Ϯ S.D. (error bars); n ϭ 3). D, immunoblotting analysis of cell lysates and exosomes from CG4 cells grown in proliferative conditions showed that PDGFR␣ is secreted. E, electron microscopy of exosomes isolated from cortices of ASA ϩ/ϩ and ASA Ϫ/Ϫ cortices. Bars, 100 nm. F and G, PDGFR␣ and PLP expression was determined by semiquantitative immunoblotting in exosomes isolated from cortices of postnatal day (P) 14 and 21 mice. Exosomal secretion of PDGFR␣ in both ASA ϩ/ϩ and ASA Ϫ/Ϫ cortices increased at postnatal day 14 and also at postnatal days 7 and 21 in ASA Ϫ/Ϫ . The level of PLP secretion was reduced in the ASA Ϫ/Ϫ cortex at both ages compared with ASA ϩ/ϩ (data shown as the mean Ϯ S.E.; n ϭ 3). ment of sulfatides in this process is demonstrated by its normalization observed after correction of the ASA deficiency and the reduction in the ratio of long and short sulfatides in treated ASA Ϫ/Ϫ NPs. Further experimentation is under way to study the molecular mechanism mediating this effect.
Our study identifies a novel cellular mechanism contributing to the regulation of the PDGFR␣ in NPs via exosomal shedding. The amount of receptor secreted by exosomes corresponds to about 3-4% of the PDGFR␣ reduction in ASA Ϫ/Ϫ NPs, which is similar to the fraction of receptor associated with DRM. These findings suggest that the fraction of receptor associated with DRM is subjected to exosome control. Low levels of PDGFR␣ in DRMs may be sufficient to interfere with critical survival/proliferation signaling in OPCs. This would weaken the capacity of the ASA Ϫ/Ϫ cultures to generate normal numbers of OPCs and OLs and explain the reduction on PDGFR␣ levels.
Exosomes are intralumenal vesicles contained in multivesicular bodies playing important roles in cell-cell communication (72)(73)(74)(75), and their shedding is a physiological relevant process. For example, the transferrin receptor and the myelin protein PLP undergo exosomal secretion during differentiation of erythrocytes and OLs, respectively (26,51,76). Sphingolipids contribute to regulating the formation of exosomes (77,78), and cholesterol and ceramides promote exosome secretion (76, 79 -81). Our experiments suggest a role for sulfatides in exosomal biogenesis. Intrinsic increases in sulfatides, such as in ASA Ϫ/Ϫ NPs, and, to a lesser extent, acute exposure to exogenous sulfatides promoted the shedding of PDGFR␣. Exosomal shedding of PDGFR␣ in glial cells has not been reported previously, but this secretory mechanism may be a relevant physiological process by which OPCs rapidly and locally regulate the concentration of receptor. In turn, this may have an immediate impact on the receptor-mediated responses to proliferation, survival, and/or differentiation.
How PDGFR␣ translocates to exosomes and is targeted for removal is still unclear. We hypothesize this process could involve changes in lipid-protein composition in membrane domains where the receptor is assembled. The changes in long versus short fatty acid sulfatides observed in DRMs in our study appear to support this. At this moment, excluding the EGFR, the possibility that other receptors are fated to exosomal secretion cannot be ruled out. Confocal microscopy studies suggested co-localization of PDGFR␣ with sulfatides in ASA ϩ/ϩ and to a lesser extent in ASA Ϫ/Ϫ NPs (data not shown). There is evidence that sulfatides are components of exosomal membranes (82). However, exosomes isolated from NPs appeared to contain little if any sulfatide (data not shown). This last observation suggests that PDGFR␣ and sulfatides might use different intracellular vesicular trafficking compartments. The reduction of PDGFR␣ from DRMs upon increased concentrations of sulfatides supports this conclusion.
In vivo studies in the ASA ϩ/ϩ cortex showed an increase in exosomal secretion of PDGFR␣ before the peak of myelination. These results provide further evidence that exosomal regulation of PDGFR␣ might be part of a larger regulatory program involved in myelination. Sulfatides may contribute to the control of the local amount of PDGFR␣ available in the OPC cell body to respond effectively to PDGF-AA during myelination. In vivo, exosomal shedding may involve other mechanisms, including axonal signals, nerve conduction, and astrocytic and microglial signals (83)(84)(85)(86)(87)(88). In this scenario, one of the events occurring when OPCs stop dividing and start myelination is the production of large quantities of sulfatides to cope with the production of the myelin membrane. Such conditions may activate the exosomal loss of PDGFR␣ from OPCs to coordinate its postmitotic cycle and myelinating program. Studies by Trajkovic et al. (50) and Bakhti et al. (51) demonstrating a fundamental role of endosomes and exosomes during OL maturation and myelination appear to support this model. Whether this mechanism is also relevant in tethering OL numbers during CNS development and remyelination is currently unknown.
In the context of the ASA deficiency, our study provides an initial understanding of how ASA deficiency and sulfatides may contribute to demyelination and remyelination defects in the human lysosomal storage disease metachromatic leukodystrophy (60). Our observations may also be extrapolated to other demyelinating diseases, such as multiple sclerosis, where demyelination could lead to the availability of myelin debris, transforming myelin-derived sulfatides into environmental toxins (31, 89 -91) that may impair remyelination. In this regard, other myelin components, such as myelin-associated glycoprotein, myelin oligodendrocyte glycoprotein, and Nogo A, are known to exert inhibitory functions, decreasing the regenerative capacity of the brain (92)(93)(94).
Our work may also extend to non-neural situations. For example, sulfatides are also expressed in non-nervous tissues, such as renal and pancreatic tissues, where they have been described as participating in the secretion of insulin (95)(96)(97), and they are elevated in a wide range of cancers, where they are thought to participate in metastasis (98 -100). In some of these diseases, sulfatides may contribute to pathology by altering the regulation of local signals via changes in DRMs and/or exosomal secretion. Because sulfatides comprise a heterogeneous population of lipids, future studies will need to determine the physiological role of the different sulfatide isoforms.