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FEBS Letters 584 (2010) 1771–1778

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Review Participation of and in glycosynapses between oligodendrocyte or membranes

Joan M. Boggs a,b,*, Wen Gao a, Jingsha Zhao c, Hyun-Joo Park a, Yuanfang Liu a, Amit Basu b

a Molecular Structure and Function Program, Research Institute, Hospital for Sick Children, Toronto, ON, Canada M5G 1X8 b Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada M5G 1L5 c Department of Chemistry, Brown University, Providence, RI 02912, United States

article info abstract

Article history: The two major of myelin, galactosylceramide (GalC) and sulfatide (SGC), interact Received 30 October 2009 with each other by trans carbohydrate–carbohydrate interactions. They face each other in the Revised 19 November 2009 apposed extracellular surfaces of the multilayered myelin sheath produced by oligodendrocytes Accepted 20 November 2009 (OLs). Multivalent galactose and sulfated galactose, in the form of GalC/SGC-containing liposomes Available online 24 November 2009 or silica nanoparticles conjugated to galactose and galactose-3-sulfate, interact with GalC and SGC Edited by Sandro Sonnino in the membrane sheets of OLs in culture. This stimulus results in transmembrane signaling, loss of the cytoskeleton and clustering of membrane domains, suggesting that GalC and SGC could par- ticipate in glycosynapses between apposed OL membranes or extracellular surfaces of mature mye- Keywords: lin. Such glycosynapses may be important for myelination and/or myelin function. raft Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Signaling Cytoskeleton Silica nanoparticle

1. Introduction Despite their abundance, these GSLs are not essential for OL development in culture or for myelin formation in vivo. Knocking Myelin is the membranous sheath that is spirally wound around out the genes encoding the enzymes that are necessary for their nerve axons (Fig. 1C) and is formed in the central nervous system synthesis, UDP-galactose: galactosyltransferase (CGT) by oligodendrocytes (OLs). The myelin membrane is rich in two and galactosylceramide 30-sulfotransferase (CST), does not prevent glycosphingolipids (GSLs), galactosylceramide (GalC) and sulfatide formation of compact myelin sheaths, which appear only slightly (the sulfated form of GalC, galactosylceramide I3-sulfate (SGC)). thinner than normal but with altered interaction of paranodal The extracellular surfaces of myelin, which face each other in the loops with the axon [2–4]. Delocalization of axolemmal proteins multilayered myelin sheath, would thus be covered with the sim- occurs around the node and paranode in these mutants, which ple sugars, galactose and sulfated galactose [1]. may be partly due to altered trafficking of myelin paranodal pro- teins to the plasma membrane [2]. However, the CGT-null mice display a severe clinical phenotype, with nerve conduction deficits, paralysis, extensive myelin vacuolation and splitting at the intra- Abbreviations: Ab, antibody; CGT, UDP-galactose:ceramide galactosyltransfer- period line, and early death [2]. The phenotype is much less severe ase; CNP, 20,30-cyclic nucleotide 30-phosphodiesterase; CST, galactosylceramide 30- sulfotransferase; DIGs, detergent-insoluble glycosphingolipid-enriched membrane in the CST knockouts, but with age, the nodal structure deterio- domains; Gal-BSA, galactose conjugated to bovine serum albumin; GalC, galacto- rates, the amount of cytoplasm in myelin increases, and myelin sylceramide; GluC, glucosylceramide; glyco-nanoparticles where glyco = Gal, SGal, vacuolar degeneration occurs [3]. Terminal differentiation of OLs Glu, Man, silica nanoparticles conjugated to galactose, galactose-3-sulfate, glucose, from the CST knockouts is enhanced in vitro and in vivo, suggesting or mannose; GPI, glycosylphosphatidylinositol; GSLs, glycosphingolipids; MAG, that SGC is a negative regulator of OL differentiation [4–6]. The myelin-associated glycoprotein; MAPK, mitogen activated protein kinase (p42 and p44, 42 and 44 kDa isoforms); MBP, myelin basic protein; MOG, myelin/oligoden- number of terminally differentiated OLs is increased in vivo and drocyte glycoprotein; PLP, proteolipid protein; OL, oligodendrocyte; SGC, sulfatide, this increase persists into adulthood in CST-null mice, due to in- sulfated form of GalC, galactosylceramide I3-sulfate creased proliferation and decreased apoptosis [7]. * Corresponding author. Address: Molecular Structure and Function Program, The myelin produced in the CGT mutant contains the hydroxy- Hospital for Sick Children, 555 University Ave., Toronto, ON, Canada M5G 1X8. Fax: +1 416 813 5022. fatty acid form of glucosylceramide (HFA-GluC) (normally absent E-mail address: [email protected] (J.M. Boggs). in OLs) instead of GalC and SGC and also increased ceramide levels.

0014-5793/$36.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2009.11.074 1772 J.M. Boggs et al. / FEBS Letters 584 (2010) 1771–1778

causes them to form more ordered membrane domains or rafts that are involved in protein trafficking [13] and/or organization of specific proteins into signaling complexes. GalC is concentrated with cholesterol, GM1, the raft marker flotillin, caveolin, glycosyl- phosphatidylinositol (GPI)-linked proteins and kinases [10–15] in low density detergent-insoluble glycosphingolipid-enriched mem- brane domains (DIGs) believed to come from membrane domains or rafts in OL and myelin membranes. These DIGs also contain SGC but are not enriched in it [11]. They also contain some myelin proteins including myelin basic protein (MBP), a peripheral mem- brane protein bound to the cytoplasmic side of the membrane, and phosphorylated MBP [12]. The GalC and SGC in these membrane domains can receive extracellular signals that are then transmitted to the cytosol.

2. Involvement of myelin GSLs in signaling

Anti-GalC and anti-SGC antibodies (Abs) have diverse effects on cultured mouse OLs and Schwann cells suggesting that they can activate GSL-enriched signaling domains in these cells. The extra- cellular signals imparted by the Abs are transmitted across the membrane to the cytoplasmic side and to the cytoskeleton (re- viewed in [16,17]) and affect OL differentiation [5,6,18]. Anti-GalC and anti-SGC Abs caused redistribution of GalC and SGC over do- mains containing MBP in OLs, and depolymerization of a lacy net- work of microtubules in the membrane sheets [16,17]. Anti-GalC Ab caused an influx of extracellular Ca2+, rapid cycling of the phospholipid polar head groups, and a decrease in phosphoryla- tion of MBP [16,17,19]. Significantly, it did not have these effects on cultured OLs from the shiverer mutant mouse, which lacks MBP, indicating that MBP is required to mediate the effects of anti-GalC Ab on OLs [19]. Anti-SGC Ab inhibited differentiation of progenitor OLs [5,6] and down-regulated gene expression in mature OLs [18] consistent with the accelerated developmental time course of cultured OLs from the CGT and CST mutants, sug- gesting that SGC is involved in signaling mechanisms that regulate differentiation [4,5,7]. Fig. 1. Sites where glycosynapses between GalC/Sulf-enriched domains in OL/ myelin membranes might occur resulting in signaling and depolymerization of the cytoskeleton. A double headed pink arrow represents glycosynapse formation 3. Transmission of transmembrane signals by GalC/SGC between two OL membranes (panels A and B), or a series of glycosynapses between interactions across apposed membranes the multilayers of the myelin sheath (panel C). (A) Glycosynapse between two different OLs – could be a signal for process retraction or cessation of growth of a membrane sheet. (B) Glycosynapse between extracellular surfaces of membranes of The effects of anti-GalC and anti-SGC Abs on cultured OLs sug- a process of the same cell wrapping around an axon (orange, labeled A) – could be a gest that there may be natural ligands that interact with these GSLs signal for elimination of cytosol and formation of closely packed (compact) myelin in signaling domains to confer signals that are transmitted across layers. (C) Series of glycosynapses between the extracellular surfaces (at intraperiod the membrane. These ligands may include axonal, OL, or extracel- line shown in light green) of compact myelin surrounding a nerve axon – could transmit extracellular or axonal signals throughout myelin layers. Major dense line lular matrix proteins, but may also be in apposed mem- where cytosolic surfaces are apposed is shown in darker green. The outer loop and branes. , including glycolipids, can adhere to each inner loop containing cytoplasm are shown in green. Reprinted from Boggs et al. [1] other by homotypic or heterotypic trans carbohydrate–carbohy- with permission from Elsevier. drate interactions between apposed membranes [20]. The interac- tions between these GSLs in GSL-enriched microdomains in apposed membranes trigger signals, perhaps as a result of non- However, elimination of HFA-GluC by knockout of the gene for covalent crosslinking or patching of the GSLs by the multivalent ar- UDP-glucose:ceramide glucosyltransferase, targeted to OLs in the ray of sugars, similar to the effect of Abs crosslinked by anti-Ig Abs. CGT mutant, has no significant effect on the phenotype, indicating These signals are transmitted across the membrane to cytosolic that the HFA-GluC does not compensate for the loss of GalC and signal transduction proteins [21,22]. Hakomori has described SGC [8]. These results suggest that galactolipids are necessary for GSL-enriched signaling domains interacting with each other across development of a normal myelin sheath that can sustain the axon apposed cell membranes as a type of ‘‘glycosynapse”, similar to the and for maintenance of compact myelin structure with age, immunological synapse [23]. although they are not necessary for the process of myelination. Like several other GSLs, the myelin GSLs, GalC and SGC, can also Galactosphingolipids may perform their role in myelin function participate in trans carbohydrate–carbohydrate interactions be- through several possible mechanisms, i.e., their ordering effect on tween apposed membranes [20,24]. We have suggested that they the lipid bilayer, their formation of rafts, and/or binding to specific may be ligands for each other and that trans interactions between ligands which trigger signaling. Galactosphingolipids have longer, them may cause similar signaling effects as the Abs [1,25].We more saturated fatty acids than phospholipids, and can also partic- have tested this hypothesis by adding phospholipid/cholesterol ipate in an intermolecular hydrogen bonding network [9], which liposomes containing GalC/SGC, or a polyvalent form of galactose J.M. Boggs et al. / FEBS Letters 584 (2010) 1771–1778 1773 conjugated to bovine serum albumin (Gal-BSA), to cultured rat OLs of the liposomes or Gal-BSA. Similar to liposomes and Gal-BSA, [25–27]. These systems would mimic an interaction which might the glyco-nanoparticles also caused clustering/redistribution of occur between GalC and SGC in apposed membranes, such as be- GalC and MBP in the OL membrane sheets (shown for Gal-nano- tween the facing pairs of extracellular surfaces of myelin, or be- particles in panels d–f of Fig. 2, compare to untreated cells in tween OLs or their processes in contact with each other (Fig. 1). panels a–c). The cells at 8 days in culture, when the glyco-nano- The GalC/SGC-containing liposomes caused redistribution of particles are added, typically consist of about 30% mature cells GalC on the extracellular side, and MBP on the cytosolic side, into with flat membrane sheets (resembling Fig. 2a–c) and are almost clusters of varying size such that the GalC domains usually overlaid completely GalC and MBP-positive, even when less mature with the MBP domains [27]. Similar clustering of GPI-linked proteins only thin processes instead of membrane sheets. However, after and of two transmembrane proteins, proteolipid protein (PLP) treatment with glyco-nanoparticles, more cells appeared less ma- and myelin/oligodendrocyte glycoprotein (MOG), occurred, sug- ture than the control cells, with many narrow processes rather gesting that the domains which cluster are membrane rafts than flat membrane sheets. In most of these less mature GalC+ [1,25,26]. Several proteins or phosphorylated proteins involved in cells, MBP staining was very low or absent, in contrast to un- signal transduction, mitogen activated protein kinase (MAPK), treated OLs. An example of this type of cell is shown in Fig. 2g– phosphorylated MBP, and some phospho-tyrosine-containing pro- i. These effects, particularly the almost complete absence of teins also clustered with MBP and GalC [26], suggesting that these MBP staining, were not commonly observed after liposome treat- rafts are membrane signaling domains. Indeed, the GalC/SGC-con- ment. Since MBP appears relatively late in OL differentiation [18], taining liposomes also caused depolymerization of microtubules its loss in the treated cells suggests that dedifferentiation may and actin filaments that form a lacy cytoskeletal network in the have occurred. The effects of the Gal, SGal, Gal/SGal and control membrane sheets, indicating that the interaction of GalC/SGC-con- nanoparticles on GalC clustering, cell morphology, and MBP stain- taining liposomes with the extracellular surface of the OL caused ing were quantified by counting the percentage of affected cells transmission of a signal across the membrane. Inhibition of a num- as described [26]. The Gal, SGal, and Gal/SGal nanoparticles all in- ber of kinases and phosphatases involved in regulation of the cyto- creased the percentage of cells with clustered or redistributed skeleton with various reagents prevented the liposome-mediated GalC, decreased the percentage of mature GalC+ cells, and de- effects on the cytoskeleton [1,28]. creased the percentage of the GalC+ cells which were also MBP- However, liposomes can affect cells either by adhering to them positive (Fig. 3). or by exchange of between the liposomes and the cells. In The Gal, SGal, or mixed Gal/SGal-nanoparticles had significantly support of the former mechanism, control liposomes without GalC more effect than Glu-, Man-, or unglycosylated-nanoparticles or SGC had much less effect on GalC redistribution than those con- (Fig. 3). The nanoparticles bearing both Gal and SGal had a signif- taining GalC and SGC. Liposomes containing only one myelin GSL, icantly greater effect on GalC redistribution than those with only either SGC or GalC, had a significantly greater effect than those Gal or SGal. Untreated mature cells have a complex cytoskeletal lacking GalC and SGC [26]. Furthermore, Gal-BSA had a similar ef- network with major veins and a lacy network of microtubules fect as the GalC-containing liposomes [26]. Similar glycoconjugates (49). The Gal/SGal nanoparticles also caused loss of the microtubu- of glucose and mannose had significantly less effect. lar network (not shown) as found for GalC/SGC-containing lipo- somes [25–27]. To determine if the receptor(s) in OLs for the glyco-nanoparti- 4. Effect of multivalent glyco-nanoparticles on OLs cles were GalC/SGC, Fab fragments of anti-GalC IgG Ab (Chem- icon) were prepared using a Pierce kit. The binding of the anti- Dendrimers or silica nanoparticles conjugated with saccharides GalC Fab fragments to OLs was confirmed using a fluorescent are other forms of multivalent presentations which have been anti-rabbit IgG F(ab0)2 specific Ab (JacksonImmuno Research used to mimic trans interactions between cell surface . Lab). For blocking of the glyco-nanoparticles, Fab fragments were Azide-functionalized silica nanoparticles can be conjugated with added to OLs at a final concentration of 2 lg/well 2 h before propargyl derivatives of sugars by copper-promoted azide-alkyne addition of the glyco-nanoparticles, the cells were washed, and cycloaddition. We have examined the effect of silica nanoparti- the glyco-nanoparticles were added. The Fab fragments under cles bearing galactose (Gal-nanoparticles), galactose-3-sulfate these conditions had no effect on the OLs after a further over- (SGal-nanoparticles), or a combination of galactose and galact- night incubation and prevented the effect of the Gal/SGal-nano- ose-3-sulfate (Gal/SGal-nanoparticles) on OLs. Propargyl b-D-3-sul- particles on GalC and MBP distribution and OL morphology (not fogalactose was synthesized by regioselective sulfonation of the shown). dibutylstannylene ketal prepared from propargyl b-D-galactose These results show that the effect of the GalC/SGC-containing [29]. Silica nanoparticles were functionalized with Gal (1.3 lmol liposomes on cultured OLs is not due to uptake or transfer of lipids galactose/mg nanoparticles, corresponding to complete coverage), between the cells and the liposomes. Moreover, a lipidic form of SGal (0.8 lmol/mg) and a mixture of Gal/SGal (0.6 lmol Gal + the sugar is not necessary for the effect; rather a multivalent form 0.5 lmol SGal/mg) (J. Zhao and A. Basu, unpublished). (Details for of the sugar, as on the surface of liposomes, or bound to a polymer nanoparticle preparation and characterization will be provided or nanoparticle, is sufficient. Finally, the effects are specific for Gal upon request.) The SGal nanoparticles were capped with propargyl and SGal. alcohol (0.4 lmol/mg particles) to block unreacted azido groups. Interactions between single sugar molecules are disrupted by Control glyco-nanoparticles contained 1.3 lmol glucose (Glu- water, but the interactions between polymeric multivalent glyco- nanoparticles) or mannose/mg particles (Man-nanoparticles). conjugates or membrane lipid domains, that present a multivalent Unglycosylated control particles were capped with propargyl alco- array of carbohydrate head groups, are stronger and persist in hol to provide a hydroxyl-terminated nanoparticle. The nanoparti- water. Furthermore, the water at the membrane surface is more or- cles were incubated with OLs at a final concentration of 2 lg/ml for dered than bulk water and less likely to hydrate the carbohydrate 6–18 h. groups [30]. The forces between multivalent glycoconjugates can The glyco-nanoparticles had a greater effect on the OLs than be measured by atomic force microscopy, surface plasmon reso- GalC/SGC-containing liposomes or Gal-BSA, with some effect seen nance and other techniques and can be strong enough to cause after 6 h and a greater effect after overnight culture. In contrast, specific adhesion of sponge cells into a multicellular organism (re- culture with the cells overnight was required to detect effects viewed in [1,25]). 1774 J.M. Boggs et al. / FEBS Letters 584 (2010) 1771–1778

Fig. 2. Confocal microscope images of OLs fixed with 4% paraformaldehyde for 10 min at room temperature and stained externally with monoclonal anti-GalC Ab (O1) (from Neuromics) (b, e, and h); then permeabilized with 0.05% saponin and stained internally with anti-MBP Ab (to peptide 82–87, from AbD Serotec) (a, d, and g). Merge is shown in (c, f, and i) (MBP, red; GalC, green). Untreated oligodendrocytes (OLs) (a–c); OLs treated overnight with 2 lg/ml Gal-nanoparticles (d–i). Panels (d–f) show MBP and GalC redistribution/clustering in a more mature cell and is typical of that caused by GalC/SGC-containing liposomes. Panels (g–i) represent a cell which looks less mature and has lost most of its MBP; this occurs frequently after nanoparticle treatment but is much less frequent with liposome treatment. OLs were cultured, treated with nanoparticles, stained with primary Abs and appropriate fluorescent second Abs, and examined by confocal microscopy as described [26]. Bar = 20 lm.

5. Receptors in OLs which interact with multivalent Gal/SGal by [1,28]. These results support our suggestion that the natural li- trans interactions gand(s) for GalC and SGC in OLs that are mimicked by anti-GalC/ SGC Abs or multivalent Gal/SGal are, or include, GalC and SGC in The receptor(s) in the OL membrane which interact with the apposed OL/myelin membranes. multivalent Gal/SGal presented by liposomes or polymers could be protein(s), but are likely to be GalC and SGC for the following 6. GalC/SGC signaling releases cytoskeletal restriction of reasons: (i) GalC and SGC can bind to each other by trans interac- membrane domains tions across apposed surfaces [20,24,25]; (ii) the effects of multiva- lent presentations of Gal/SGal on OLs resemble the effects of anti- Jasplakinolide, a reagent which stabilizes actin filaments, inhib- GalC/SGC Abs reported by Dyer and Benjamins [16,17] in many re- ited liposome-induced redistribution of all the membrane constit- spects; (iii) Fab fragments of anti-GalC IgG Ab, which had no effects uents which were clustered in its absence, including GalC on the on OLs themselves, prevented the effects of the Gal/SGal-nanopar- extracellular surface [26]. It also prevented depolymerization of ticles on GalC and MBP distribution and OL morphology (not the microtubules. This result indicates that depolymerization of shown); (iv) the multivalent Gal/SGal had no effect on the cyto- the actin filaments is required both for redistribution of the mem- skeleton of astrocytes that were also present in the culture, and brane constituents and for depolymerization of the microtubules. that lack these two GSLs, indicating a specific effect on GalC/SGC- Thus, it is an early event in the transmembrane signaling mediated containing OLs [25,27]; (v) inhibition of GSL synthesis by treat- by multivalent Gal/SGal. The fact that it also prevented clustering ment of OLs with fumonisin B1 prevented the effect of liposomal of GalC on the extracellular surface suggests that GalC is somehow GalC/SGC on MBP redistribution in the GalC/SGC-negative OLs linked to elements on the cytoplasmic side and/or that it is not J.M. Boggs et al. / FEBS Letters 584 (2010) 1771–1778 1775

to the plasma membrane in OLs [34,35]. Low density DIGs isolated from OLs and myelin also contained actin and tubulin [11,36,37] in addition to MBP, indicating that rafts may be linked to the mem- brane skeleton in OLs via MBP, in addition to other proteins (Fig. 4). These results also suggest that MBP may be linked to cyto- skeletal proteins even in myelin. DIGs from myelin also contain the radial component, which is made up of tight junctions that may control the ionic content of the extracellular space in myelin [1,38].

8. Role of glycosynapses in OLs or myelin

We have suggested that GalC/SGC-enriched microdomains in the OL/myelin membrane interact with each other in glycosynaps- es between apposed membranes of OL processes in contact with each other, or between the extracellular surfaces of compact mye- lin (Fig. 1). If OLs are in contact at high densities, or if OL processes contact an already myelinated axon, this contact might cause pro- cess retraction involving disruption of the cytoskeleton (Fig. 1A). A signal applied to SGC via a glycosynapse might be expected to in- hibit differentiation and process extension [4] as observed with Fig. 3. Effect of glyco-nanoparticles (2 lg/ml) on OLs after overnight incubation. anti-SGC Ab [5,6,18]. The increased number and decreased apopto- Percentages of cells with clustered GalC, mature cells, and MBP+ cells are shown sis of OLs in the CST-null mouse [7] may be due to a failure of con- after no treatment (black) or after treatment with particles bearing Gal (red), SGal tact inhibition normally mediated by GalC/SGC interactions. (green), Gal/SGal (yellow), Glu (dark blue), Man (magenta), unglycosylated Dynamic regulation of the cytoskeleton is necessary for various (turquoise). Data represent means ± S.D. of 3–6 experiments. The percentages of affected cells (clustered GalC, mature cells, and MBP+ (of GalC+ cells) cells) after stages of myelination [1,39]. When mature myelinating OLs ini- treatment with Gal/SGal-nanoparticles are significantly different from those treated tially ensheath axons, the first few layers of membrane around with unglycosylated-nanoparticles, P < 0.001. The percentage of cells with clustered the axon contain cytosol [40]. Disruption of the cytoskeleton in GalC after treatment with Gal/SGal-nanoparticles is significantly different from that these layers is necessary for the cytoplasmic surfaces to adhere after treatment with SGal- or Gal-nanoparticles (P 6 0.05). Statistical significance and create compact myelin. GalC–SGC interactions could occur as determined by Student’s t-test. the membrane sheets wrap around the nerve axon allowing GSLs in apposed surfaces to come into contact at least transiently and/ individual molecules that redistribute, but rather entire membrane or in localized domains and confer a signal for compaction domains/rafts that redistribute and coalesce (Fig. 4). (Fig. 1B). In CST-null mice, the processes which myelinate are Since actin depolymerization is an early event following inter- thicker and retain more cytoplasm than in wild-type mice [7], per- action of multivalent Gal/SGal with the OL membrane, this interac- haps due to retention of the cytoskeleton in the absence of GalC/ tion must first cause transmission of a signal across the membrane Sulf interactions. Formation of compact myelin requires close which affects actin. This initial signal may be Ca2+ entry, as found apposition between each pair of facing extracellular surfaces, and when anti-GalC Ab was added to OLs [16,17], or a mechanical sig- between each pair of facing cytoplasmic surfaces, which may be nal. Depolymerization of the cytoskeleton then allows redistribu- promoted by protein and GSL clustering. Fitzner et al. [14] have tion and coalescence of microdomains enriched in GalC, MBP, shown that co-culture of OLs with neurons induced GalC ordering and the other membrane constituents examined. This sequence in OL membranes, which was probably due to GalC clustering, and of events suggests that the cytoskeleton restricts lateral diffusion increased MBP distribution into DIGs. Thus, a neuronal signal of these membrane constituents, either by binding to them or by inducing OL processes to ensheath the axon could be followed by binding to other transmembrane proteins. This is consistent with GalC/SGC contact between the apposed membranes, depolymer- the membrane skeleton fence or picket fence model [31] and with ization of the cytoskeleton, and protein and GSL clustering, leading studies indicating that lipids and transmembrane proteins undergo to elimination of cytosol and adhesion of the cytosolic surfaces. hop diffusion in compartmentalized membrane domains of 50– Signaling resulting from trans GalC–SGC interactions may also 200 nm [32,33]. Upon depolymerization of the cytoskeleton, these occur in the mature myelin sheath (Fig. 1C). Although X-ray dif- domains are able to redistribute and coalesce into large clusters fraction and electron microscopy indicate that the static separation (Fig. 4). Non-covalent crosslinking or patching of the small mem- of the extracellular surfaces of compact myelin is too great for the brane domains/rafts restrained within these compartments by carbohydrate head groups of these GSLs to be in contact [41],we the multivalent Gal/SGal may facilitate this redistribution. have argued that they may come into transient contact under some conditions that cause protein clustering, such as increased extra- 2+ 7. Role of MBP in transmission of GalC/SGC-mediated signal cellular Ca concentration [1,25]. Communication between the myelin sheath and the axon may regulate both axonal and myelin MBP on the cytosolic side may play an important role in trans- function and provide trophic support to the axon [42]. This trophic mission of the GalC/SGC-mediated signal to the cytoskeleton. Ear- support by the myelin sheath may compensate for its shielding of lier studies by Dyer et al. showed that anti-GalC Ab did not cause the axon from extracellular metabolic support. It may be necessary effects in OLs from the shiverer mutant mouse, which lacks MBP to prevent neurodegeneration, since neurodegeneration occurs in [19]. Suppression of MBP synthesis in normal rat OLs using MBP mutant mice in which one of several myelin proteins, such as siRNA significantly inhibited the effect of GalC/SGC-containing lip- PLP, 20,30-cyclic nucleotide 30-phosphodiesterase (CNP), or mye- osomes on GalC redistribution in the MBP-negative OLs [28] and on lin-associated glycoprotein (MAG), is eliminated, even though an the cytoskeleton (Boggs and Gao, unpublished). MBP binds to and apparently normal myelin sheath is formed [43]. The myelin assembles actin filaments and microtubules and binds actin and sheath has recently been shown to produce ATP through genera- microtubules to a lipid bilayer, and it may tether the cytoskeleton tion of a proton gradient across the lamellae, and has been postu- 1776 J.M. Boggs et al. / FEBS Letters 584 (2010) 1771–1778

Fig. 4. Effects of multivalent Gal/SGal on OL membrane sheets. GalC and SGC-enriched membrane domains (rafts) in OL membrane sheets also contain MBP, a peripheral membrane protein on the cytoplasmic side, and are linked to the membrane skeleton (made up of linked blue spheres) via MBP [34,35], transmembrane proteins and/or other membrane-actin binding proteins. Some transmembrane proteins (blue oblongs) bound to the membrane skeleton serve as picket fences, according to the hypothesis of Kusumi and co-workers [31–33], that restrict lateral diffusion of both lipids and proteins in the membrane domains. Upper panel – binding of multivalent Gal/SGal (GalC/ SGC-containing liposomes or glyco-nanoparticles conjugated to Gal and SGal, depicted by large purple spheres bearing Gal (lavender hexagon) and SGal (lavender hexagon with small yellow sphere)), crosslinks GalC and SGC molecules in the membrane domains, and triggers an initial signal, possibly Ca2+ entry, which causes dissociation of actin filaments and microtubules and their depolymerization. Lower panel – loss of the membrane skeleton permits lateral diffusion of the membrane domains so that they coalesce into larger clusters. The membrane domains may contain a number of other transmembrane proteins such as PLP and MOG, and signaling proteins such as MAPK (not shown), since they redistribute together with GalC, SGC, and MBP [26]. MBP on the cytoplasmic side may be linked to GalC/SGC on the extracellular side via one of these transmembrane proteins, since MBP is influenced by the GalC/SGC crosslinking and is necessary for transmission of the extracellular signal to the cytoskeleton [19,1]. The head groups of the lipids are depicted as GalC (green) SGC (purple), (yellow), phospholipids (PL) (pink); cholesterol (red rod). Similar interaction between GalC/ SGC-enriched domains in apposed OL or myelin membranes is postulated to create a glycosynapse and have a similar signaling effect.

lated to be a site of oxygen absorption and aerobic metabolism for the caliber and shape of the axon with age [3], suggesting that the axons [44]. Participation of transient GalC and SGC interactions the lack of SGC decreased signaling from myelin to the axon. between the apposed extracellular surfaces of mature myelin might allow transmission of signals throughout the myelin sheath 9. Altered GalC/SGC interactions in demyelinating disease regulating such metabolic activity, and thus facilitate myelin–axo- nal communication and trophic support of the axon. In support of Interestingly, IgM Ab to GalC or SGC, produced by hybridoma this conclusion, deletion of SGC in the CST-null mouse decreased cells implanted in the spinal cord in vivo, or in an in vitro myelinat- J.M. Boggs et al. / FEBS Letters 584 (2010) 1771–1778 1777 ing culture, resulted in formation of myelin with a large space be- [15] Dupree, J.L. and Pomicter, A.D. 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