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Progressive axonal degeneration follows demyelination in many neurological diseases, including multiple sclerosis and inherited demyelinating neuropathies, such as Charcot-Marie-Tooth disease. One glial molecule, the myelin-associated glycoprotein (MAG), located in the adaxonal plasmalemma of myelin-producing cells, is known to signal to the axon and to modulate axonal caliber through phosphorylation of axonal neurofilament proteins. This report establishes for the first time that MAG also promotes resistance to axonal injury and prevents axonal degeneration both in cell culture and in vivo. This effect on axonal stability depends on the RGD domain around arginine 118 in the extracellular portion of MAG, but it is independent of Nogo signaling in the axon. Exploiting this pathway may lead to therapeutic strategies for neurological diseases characterized by axonal loss.
A fundamental problem in neurology is the tendency for demyelinated nerve fibers to undergo degeneration and loss. In diseases of presumed immune pathogenesis such as multiple sclerosis (MS) in the central nervous system (CNS) and the Guillain-Barre Syndrome in the peripheral nervous system (PNS), local inflammation undoubtedly plays a role. There is now a wealth of evidence that demyelination in itself also can contribute to altered axonal transport, axonal degeneration and loss (de Waegh et al., 1992; Trapp et al., 1998; Coleman and Perry, 2002; Ciccarelli et al., 2003; Oh et al., 2004). Examples include heritable demyelinating diseases of both the CNS and PNS. Many of the genetic defects in these diseases are abnormalities in genes encoding intrinsic myelin proteins and not expressed in axons. The clinical manifestations are often due to progressive, distally predominant axonal degeneration (Berciano et al., 2000; Krajewski et al., 2000).
The myelin-associated glycoprotein (MAG) is a component of all myelinated internodes, whether formed by oligodendrocytes in the CNS or by Schwann cells in the PNS. MAG is distinctively located in the adaxonal plasmalemma that apposes the axon as well as the paranodal loops, Schmidt- Lanterman incisures, and mesaxons (Trapp and Quarles, 1982). The normal role of MAG is poorly understood. MAG is not necessary for myelination, and myelin sheaths of MAG knockout mice (MAG-/-) are largely normal (Yin et al., 1998). Its distribution has prompted the hypothesis that MAG prevents compaction of myelin membranes and contributes to the uniform intermembranous distance characteristic of the periaxonal space (Trapp and Quarles, 1982). MAG is known to signal to the axon, locally influencing the phosphorylation of axonal neurofilaments immediately beneath MAG-bearing membranes because of reduced interfilament spacing (Hsieh et al., 1994; Dashiell et al., 2002). Myelinated axons of MAG-/- mice have smaller diameters than normal as a result of hypophosphorylation of the neurofilament proteins NF-H and NF-M (Garcia et al., 2003; Rao et al., 2003).
Much of the published research on MAG has focused on its ability to inhibit axonal elongation during regeneration. Multiple axonal receptors have been proposed to mediate MAG-induced growth cone collapse. One is a multicomponent complex consisting of a ligand-binding Nogo-66 receptor (NgR) and two transmembrane coreceptors, LINGO-1 and either p75 or TROY (Fournier et al., 2003; Mi et al., 2004). A second is proposed to be gangliosides GD1a and GT1b. Interaction of MAG with axons involves at least two recognition sites: one around arginine 118 (R118) in Ig domain 1 and second in Ig domains 4 and 5 (Kelm et al., 1994; Tang et al., 1997; Cao et al., 2007). While Ig domains 4 and 5 are believed to be important for the interaction with NgR, the R118 binding site is thought to involve in interactions with gangliosides. Its other roles the R118 binding site remain unclear.
MAG-/- mice develop axonal loss in the CNS and PNS (Yin et al., 1998; Pan et al., 2005), suggesting that MAG may influence axonal maintenance. This prompted us to hypothesize that, in addition to MAG's well-described inhibitory effects on axonal outgrowth, an additional role of MAG in the normal nervous system may be to promote stability and survival of myelinated axons. In this study we have asked whether MAG has protective effects on axons and if so, what region of the MAG molecule is involved and whether these effects depend on NgR.
The following were obtained commercially: acrylamide (Sigma-Aldrich, St. Louis, MO); Neurobasal medium (Invitrogen, Carlsbad, CA); fetal bovine serum (FBS) (Hyclone, Logan, UT); L-glutamine (Invitrogen, Carlsbad, CA); B27-serum free supplement (Invitrogen, Carlsbad, CA); nerve growth factor (NGF) (Sigma-Aldrich, St. Louis, MO); trypsin (Invitrogen, Carlsbad, Ca); collagenase I (Worthington, Lakewood, NJ); β- III tubulin (Promega, G7121); anti-MAG (R & D Systems, AF538); anti—Fc (R & D Systems, AF2049); anti-tyrosinated tubulin (Sigma-Aldrich, T9028); anti-detyrosinated tubulin (Chemicon, AB3201); MAG-Fc (R & D Systems, 538-MG); Fc (R & D Systems, AF2049); recombinant active MMP-7 (Calbiochem/EMD Biosciences , Cat. No. 444270). Mutant proteins MAG-Fc and OMgp were provided by Dr. Guo-Li Ming.
MAG knockout founder mice, kindly provided by Dr. John Roder, University of Toronto, Ontario, Canada, were constructed by disruption of exon 5 of the MAG gene as previously reported (Ng et al., 1996). The strain provided (identical to that available from the Jackson Laboratory, Bar Harbor, ME) was on a C57BL/6, 129 inbred strains and CD1 Random bred strain. To enhance comparisons between mutant strains, mutant mice were repeatedly back-crossed onto a C57BL/6 background to >99% strain purity (Pan et al., 2005).
NgR1 knockout and wild-type liter-mate mice, kindly provided by Dr. Stephen Strittmatter, Yale University School of Medicine, New Haven, CT, were constructed by disruption of exon 2 of the NgR gene as previously reported (Kim et al., 2004). The strain provided was on a C57BL/6 background.
Mice were anesthetized with chloral hydrate and perfused through the ascending aorta with freshly prepared 4% paraformaldehyde in 0.1 M sodium phosphate (pH-7.4). The C-5 spinal cord, sciatic nerves, and distal tibial nerves were harvested from groups of mice at 6, 12, and 15 months of age. Five mice were used for each group. The tissues were further fixed in 4% paraformaldehyde / 3% glutaraldehyde in Sorenson's buffer overnight at 4°C, post-fixed in OsO4, and embedded in Epon-Araldite resin.
Cross sections (1-μm thick) were stained with toluidine blue for analysis under light microscopy with a 60X or 100X oil immersion objective lenses using a stereotactic imaging software (Stereo Investigator version 5). Every myelinated fiber in easily defined regions of the CNS and PNS was counted (Supplemental Fig 1). The resulting data were free of counting biases that could affect both random and systematic (stereologic) schemes of number of axon counting. Actively degenerating fibers were defined as fibers at different stages of Wallerian-like degeneration, such as myelin figures and ovoids. Results from each studied group were compared by the two-tailed Students t-test. p<0.05 was considered significant.
Groups of five 6-week-old male mice were treated with acrylamide by adding acrylamide to the drinking water at 400 ppm. Control mice drank regular water. Five mice were housed in each plastic cage throughout the experimental period. Experimental procedures followed the principles in the “Use of Animals in Toxicology” and NIH guidelines (“Guide for the Care and Use of Laboratory Animals,” NIH Publication No. 86-23, 1985).
Groups of five mice were trained one week prior to acrylamide treatment on 3 consecutive days by performing the Rotarod at a constant speed of 4 rotation per minute (rpm) on day 1 and 8 rpm on day 2 and 3 for 3 minutes (pre-training), followed by an accelerating protocol in which the speed of rotation was initially set at 4 rpm and accelerated an additional 4 rpm every 30seconds to a maximum of 40 rpm. Three trials were performed on each mouse on each of 3 consecutive days with approximately 30 minutes rest between trials. Following initiation of acrylamide treatment, the mice were further trained for another 3 consecutive days. Two weeks after acrylamide intoxication, mice were again pre-trained and tested for 3 days, 3 trials per day. Unlike untreated mice, most MAGKO mice fell off the rod even during pre-training (mice were not returned to the rod more than twice during pre-training).
To analyze data, we recorded the speed and duration during which the mouse stayed on the rod. Results of all 3 trials on day 3 of testing were pooled to generate a mean and SEM. (n= 15). Results were compared by the two-tailed Students t-test. Values of p<0.05 were considered significant. The tester was blinded to the treatment. No mice or values were excluded.
Nerve conduction studies were performed on five wild-type and five MAG knockout mice at 6-8 weeks of age. The mice were anesthetized with isofluorane using a nose cone. Body temperature was maintained on a warm blanket to keep the surface temperature between 32° and 38°C prior to the taking of measurements. All compound motor action potentials (CMAP) measurements were performed with a PowerLab signal acquisition setup (ADInstruments). The CMAPs were measured by stimulation with subdermal needle electrodes placed near the sciatic nerve at the sciatic notch. Recording electrodes were placed in the tibial nerve-innervated intrinsic foot muscles in the plantar surface. Recordings were made with supramaximal stimulation. We determined the latencies, negative peak amplitudes, and durations of the sciatic compound muscle action potentials as well as the F-wave latencies and durations. The distance between stimulation sites, determined by calipers, was also used to calculate conduction velocities. CMAP measurements were obtained at the initiation of acrylamide intoxication and 2-1/2 weeks after intoxication.
P4-5 dorsal root ganglia (DRG) were removed from Sprague-Dawley rat pups and maintained in neurobasal medium containing 2M L-glutamine, 2% B27-serum free supplement and 50-100ng/ml nerve growth factor (NGF). Explant cultures were allowed to grow to a mature and maintenance state for 5 days, creating a lush outgrowth of neurites. This method of allowing neuritic outgrowth extension to proceed before addition of MAG and/or a neurotoxin test their effect on established neurites as opposed to their effect on primary neuritic outgrowth. The neurons were examined under phase contrast microscopy, and the average neurite length were quantified by using IMAGE J, a public domain image processing program (http://rsb.info.nih.gov/ij/). The axonal lengths of all axons were measured from the explant's border to the tips of intact axons. An average of all axonal lengths for each explant was then calculated. At least 8-10 explants were used for each test conditions.
In experiments involving MAG-CHO cells, DRG explants were allowed to grow to a mature and maintenance state for 5 days. MAG-CHO cells were then added to the culture and allowed to grow for additional 48 hours before the effect of MAG-CHO cells on axonal protection was examined.
A tetracycline repressible MAG system was generated in Chinese hamster ovary (CHO) cells as described previously (Milward et al., 2008). Several clones were derived and simultaneously assessed for expression of MAG and DsRed2, tetracycline repressibility of MAG and membrane-bound expression of MAG. Addition of the tetracycline analog doxycycline at 1 μg/ml caused complete inhibition of MAG expression. In the present studies, MAG was allowed expressed throughout the experiments.
Results from each studied group were compared by the two-tailed Students t-test. p<0.05 was considered significant.
P4-5 dorsal root ganglia (DRG) and cortical neurons were removed from 4 animals and incubated with 0.25% trypsin. The digestion solution for post-natal DRG neurons also included 0.3% collagenase I. After 30 minutes, digestion was stopped with 10% FBS containing L-15 medium, and cells were plated at 10,000/cells per well. Cultures were maintained in neurobasal medium containing 1% FBS, 2M L-glutamine, 2% B27-serum free supplement and 50-100ng/ml nerve growth factor (NGF). Neuronal cultures were allowed to grow to a mature and maintenance state for 5-7 days, creating a lush outgrowth of neurites. The average neurite lengths of 50 neurons immunostained for class III β-tubulin were quantified under fluorescent microscopy using Image Lab. Each experiment was performed in triplicate. Results from each studied group were compared by the two-tailed Students t-test. p<0.05 was considered significant.
Dissociated DRG and cortical neurons were plated onto collagen-coated tissue culture dishes in the middle of a three-compartment chamber. Compartmentalized cultures were prepared as previously described (Campenot, 1982). Cells were maintained for the initial 2-7 days in growth medium containing cytosine arabinoside (10 uM) to eliminate Schwann cells and non neuronal, dividing cells. After growing for 7 days, axons from the central chamber extended into the side chambers. The central compartment contained the cell bodies and proximal axons (M), while the side compartments contained the distal axonal processes and axon terminals (D). Treatment mediums were added to the side chambers, allowing selective delivery of the stimuli to axons. The average axonal lengths of 50 neurons immunostained for class III β-tubulin were quantified under fluorescent microscopy using Image Lab. Axonal length was measured from the edge of the chamber to the intact axonal tip. Each experiment was performed in 5-8 culture plates. Results from each studied group were compared by Students t-test. Values of p<0.05 were considered significant.
Mutation on MAG (from RGD to KGE) was generated by site directed mutagenesis (Stratagene) as described by the manufacturer. Mutant MAG-Fc with KGE domain was then transfected into 293 Ebna cells. The proteins were collected from the media and affinity purified using protein A sepharose. Wild-type MAG-Fc with RGD domain also were expressed and purified in a similar manner.
MAG was extracted from purified myelin membranes using mild detergent and was adsorbed to culture surfaces as described (Mehta et al., 2007). Briefly, myelin was purified from brains freshly dissected from adult Sprague-Dawley rats or adult wild-type or MAG-null mice and stored at -70°C prior to use. Myelin membranes were suspended at 1 mg protein/ml in extraction buffer (0.2 M sodium phosphate buffer (pH 6.8), 0.1 M Na2SO4, 1 mM EDTA, 1 mM DTT, protease inhibitor mixture (Sigma), and 1% octylglucoside), incubated at 4 °C for 16 hours with gentle agitation, then centrifuged at 100,000 × g for 1 hours at 4°C. The supernatant was collected and diluted with an equal volume of detergent-free buffer and an aliquot (50 μl) was added to each well of a PDL-coated 96-well plate. After 4 hours at ambient temperature, the plate was washed with Dulbecco's phosphate-buffered saline (PBS) and then with the culture medium appropriate to the cell type prior to plating freshly prepared cells. DRG and cortical neurons were grown on the substrate as described previously (Mehta et al., 2007).
After 48 hours cultures were washed with PBS, fixed overnight with 2% paraformaldehyde in PBS, then permeabilized using 0.1% Triton X-100 in PBS. DRGN were immunostained with anti-neuronal class III β-tubulin monoclonal antibody (TUJ1, 1:2000, Covance, Berkeley, CA) followed by Cy3-conjugated anti-mouse IgG (1:200; Jackson ImmunoResearch Laboratories, West Grove, PA). After washing, multiple random fields were captured for image analysis using a Nikon TE300 epifluorescent microscope fitted with a Photometrics CoolSNAP HQ2 camera (Roper Scientific, Duluth, GA).
DRG neurons on control substrata extended long, thick neurites when grown in control conditions. In contrast, DRG neuronal cultures grown in the presence of vincristine (without myelin substrata) have neurites that progressively degenerate distally where they become fragmented, comparatively thinner, and morphologically distinguishable from neurites in cultures without vincristine. To differentiate the integrity of neurites, we developed an image analysis protocol using NIS-Elements software (Nikon, Melville, NY) to detect the intact, non-fragmented axons. The protocol selected neurites that were >150 μm long, and subtracted out the portion of neurites that are fasciculated. Neuritic lengths were summed and divided by the total number of DRG neuronal cell bodies to provide a single value for the presence of healthy unfasciculated neurites.
For each experimental condition 4-5 random images from each of 5-10 independent wells from an average of 3 independent experiments were analyzed. Data are presented as the mean ± standard error of the mean. Results were compared using the two-tailed Students t-test. Values of p<0.05 were considered significant.
Cell monolayers were scraped and lysed in a detergent-containing buffer (10 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 2 mM sodium orthovanadate, 10 mM NaF, 10 mM sodium pyrophosphate, 1% IGEPAL, 1 mM phenylmethylsulfonyl fluoride) with protease inhibitor cocktail (Roche, Mannheim, Germany). Total protein (10 μg) was loaded on 4-12% Tris/Glycine gradient gels and transfered to nitrocellulose membranes (Novex, San Diego, CA, USA). Membranes were blocked in 10% bovine serum albumin (BSA) in Tris-buffered saline (TBS)/0.05% Tween-20 (TBS-T) for 1 hour at room temperature, and then incubated with the primary antibodies and their appropriate peroxidase-conjugated secondary antibodies for varying times. Blots were developed using enhanced chemiluminescence (ECL) (NEN Life Science Products, Boston, MA, USA). Antibodies for specific proteins are listed in the Reagents section.
Skin tissue was fixed for 12-18 h in 2% paraformaldehyde/lysine/periodate fixative followed by cryoprotection. The tissue were sectioned with a sliding microtome into 50-mM thick free-floating sections. Sections were stained with monoclonal antibodies to neurofilament (NF160 1:200, Chemicon) and developed with chromogens as previously described (Ebenezer et al., 2007).
We confirmed that MAG-/- mice underwent progressive axonal degeneration within the CNS and PNS (Yin et al., 1998; Pan et al., 2005). The numbers of intact nerve fibers and those undergoing degeneration were enumerated in the rostral medial dorsal columns of the spinal cord at the C5 level (Supplemental Fig. S1). This site contains the preterminal regions of the central processes of neurons located in the sacral and lumbar dorsal root ganglia. These axons are thus among the longest axons in the body. In wild-type mice there were very few actively degenerating fibers and no detectable axonal loss between 6 and 15 months of age (Fig. 1a(i)). In contrast, in the rostral medial dorsal columns of MAG knockout mice there was a nearly constant level of ongoing axonal degeneration involving 0.46% of fibers at any time between 6 and 15 months, resulting in progressive decrease in axonal numbers, cumulating in a 28% reduction by 15 months (Fig. 1a(ii)). A similar progressive loss of axons was found in the sciatic and the tibial nerves of MAG-/- mice (Fig. 1b, Supplemental Fig. S2).
To evaluate whether the absence of MAG results in increased vulnerability to axonal degeneration from additional stresses such as neurotoxins, we administered acrylamide, a well characterized toxin causing axonal “dying back” or degeneration without lymphocytic inflammatory response (Schaumburg et al., 1974; Ko et al., 2000) to wild-type and MAG-/- mice. When wild-type mice at 6 weeks of age were exposed to acrylamide for 3 weeks, there was mild axonal swellings and scattered, distally predominant axonal degeneration in the skin, sciatic nerve, and C-5 spinal cord (Fig. 1c(i), Supplemental Fig. S3). On gross observation, there was only mild gait unsteadiness. When challenged with the Rotarod test, acrylamide intoxicated wild-type mice had mild difficulty in maintaining their balance on the rotating rods and had a modest decline in retention time on the Rotarod in comparison with untreated mice (Fig. 1d). This was accompanied by a small decrease in amplitudes of CMAP on electrophysiological testing (Fig.1 e). Quantitative morphological analysis of the sciatic nerve and medial dorsal column of C-5 spinal cord revealed a small loss in the total number of axons following acrylamide exposure (Fig. 1f). In contrast, MAG knockout mice exposed to acrylamide showed markedly impaired gait with marked sensory ataxia and spreading of the toes of the hind limbs (Supplemental Video 1). MAG knockout mice had lower retention time on a Rotarod in comparison to wild-type controls. Upon acrylamide exposure, MAG knockout mice developed more rapid and severe motor impairment. Most MAG knockout mice fell off the rod even during pre-training and had profound decline in the retention time on the Rotarod (Fig. 1d). There was a 36% decrease in amplitudes of CMAP (Fig. 1e). Likewise, morphological analysis showed significant axonal degeneration with almost 20% loss in total number of axons in the sciatic nerve and medial dorsal column of C-5 spinal cord (Fig. 1f, Supplemental Fig. S3). This was accompanied by numerous axons in the skin with profound swelling, beading and segmentation consistent with predegenerative changes of Aβ fibers (Fig. 1c(ii)) (Schaumburg et al., 1974; Hsieh et al., 1996; Ebenezer et al., 2007).
To test whether the absence of MAG also results in increased vulnerability to axonal degeneration from CNS inflammation, we employed a widely used animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). EAE was induced by immunizing wild-type and MAG knockout mice with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55. Wild-type mice induced with MOG-EAE exhibited severe axonal loss in the rostral medial column at 4 weeks. MAG-/- mice showed 52% more axonal loss at 4 weeks following MOG-EAE induction as compared to wild type mice at the same time point (Fig. 1g). Rotarod testing was not performed on EAE induced mice; both EAE-MAG-/- and EAE-wild-type mice exhibited motor impairment sufficiently severe that Rotarod testing was not informative.
In order to further elucidate the effects of MAG on axons, we established postnatal (P5) primary rat dorsal root ganglion cultures. We confirmed that the addition of soluble MAG-Fc to the cultures resulted in phosphorylation of NF-H (data not shown) (Dashiell et al., 2002). We next asked if MAG affected microtubule stability. Recently assembled and cold-labile microtubules contain greater amounts of tyrosinated tubulin than do older, more long-lived ones and cold stable microtubules (Baas and Black, 1990). For this reason, the ratio of detyrosinated tubulin to tyrosinated tubulin is considered a marker of microtubule stability. Following the addition of MAG-Fc to rat cerebrocortical cultures, we noted increased levels of detyrosinated tubulin and decreased levels of tyrosinated tubulin (Fig. 2a). These data suggest that MAG increases microtubule stability.
To ensure that the effects were local effects on the axonal segments exposed to MAG, we utilized Campenot chambers (Campenot, 1992). These chambers use a grease seal to produce a physical, water-tight separation of distal axons from neuronal cell bodies. When soluble MAG (MAG-Fc) was added to the distal axonal compartment of these cultures, there was a decrease in subsequent axonal longitudinal growth (Fig. 2b), an observation consistent with the existing literature (DeBellard et al., 1996). To ask if these stabilized axons were more resistant than untreated axons to injury we added vincristine (VIN), a chemotherapeutic agent with well-characterized properties of axonal toxicity, to the distal chamber of postnatal DRG cultures. In cultures not treated with MAG-Fc, VIN produced a marked reduction in axonal length compared to baseline, and there was morphological evidence of axonal degeneration. When MAG-Fc was co-administered with vincristine to the distal axonal chamber, vincristine-induced axonal degeneration was largely prevented (Fig. 2b). Hence, this suggested that while MAG-Fc may discourage longitudinal axonal growth, it simultaneously promotes axonal stability and resistance to axonal degeneration.
MAG-Fc similarly conferred axonal protection from vincristine in post-natal (P5) DRG explant cultures (Fig. 2c, ,2d)2d) and dissociated embryonic (E15) cerebrocortical cultures (Supplemental Fig. S4). A dose-response curve of MAG-Fc axon protective effect against vincristine is shown in Figure 2.e. This axonoprotective effect was not restricted to vincristine, as MAG also prevented axonal degeneration in response to acrylamide, granzyme B, and supernatants from activated cytotoxic T cells (Fig. 2f).
To ensure that the effects were not limited to this soluble MAG-Fc fusion protein, the axonal protective ability of different forms of MAG was investigated. After allowing post-natal DRG explants to reach a mature and maintenance state, they were co-cultured with MAG-CHO cells expressing a high level of membrane associated MAG (Milward et al., 2008). In the presence of MAG-CHO cells, vincristine-induced axonal degeneration was largely prevented (Fig. 2g). To explore the functional domain of MAG's protection, MAG was cleaved at a juxtamembrane position on MAG-CHO cells by MMP-7 to release an intact extracellular fragment (Milward et al., 2008). When the extracellular MAG fragments were removed with regular interval washes, the ability of MAG-CHO cells to protect against vincristine was lost (Fig. 2g). When the medium containing the extracellular MAG fragments was placed on neurons grown without MAG-CHO cells, vincristine-induced axonal degeneration was now largely prevented (Fig. 2g). This suggests that like the growth inhibitory effects, axonal protection depends on the extracellular Ig domains of MAG for the relevant functional domain for axonal protection (DeBellard et al., 1996).
Finally, post-natal DRG neurons were grown on poly-D-lysine matrix alone or with full length native MAG detergent-extracted from adult rat brain myelin (Mehta et al., 2007). Since extension of axons of DRG neurons is inhibited in an NgR-mediated fashion when grown on detergent extracted myelin (Mehta et al., 2007), phosphatidylinositol-phospholipase C (PI-PLC) was added to the cultures to overcome MAG-mediated inhibition in order to study whether MAG could protect axons (Fournier et al., 2001; Liu et al., 2002; Mehta et al., 2007). When DRG neurons were grown on detergent extracts of myelin, the vincristine-induced axonal degeneration was largely abolished (Fig. 3a, ,3c).3c). Anti-MAG antibody reversed the protection against vincristine (Figs. 3b, ,3c,3c, similar results in cortical neurons are not shown). Myelin extract from MAG-/- also failed to provide axonal protection (data not shown). Taken together, our in vitro and in vivo data thus suggest that the axonal protective effect of MAG is not restricted to a particular form of MAG, neuronal age, or neuronal cell type (CNS or PNS).
We next asked whether a Nogo receptor (NgR), a member of a family of glycosylphosphatidylinositol (GPI)-linked receptors, was necessary for MAG-induced protection from axonal degeneration. In contrast to the axonal degeneration and loss seen in the MAG -/- mice, NgR1 -/- mice exhibited no actively degenerating fibers in the rostral medial dorsal column of the spinal cord, in the sciatic nerves (Fig. 4a), or in the tibial nerves (Supplementary Fig. S4). Moreover, in cerebrocortical and DRG neuronal cultures, removal of GPI-linked molecules like NgR from the cell surface by incubation with PI-PLC had no effect on the ability of MAG to prevent vincristine-induced neuritic degeneration (Fig. 4b) (Fournier et al., 2001; Domeniconi et al., 2002; Liu et al., 2002). Similarly, the activation of the Nogo signaling pathway with Nogo-66 or OMgp peptides, which non-selectively interact with various NgR receptor types, did not provide axonal protection against vincristine (Fig. 4b). These data suggest that an axonal receptor other than NgR mediates MAG-induced axonal protection.
The interaction of MAG with axons previously had been proposed to include a second binding site at arginine 118 (R118) in first Ig domain of MAG, which is not required for NgR-mediated inhibition of axonal elongation during regeneration (Kelm et al., 1994; Tang et al., 1997; Cao et al., 2007). In our system, an R118-mutated MAG failed to prevent axonal degeneration caused by vincristine (Fig 4c). This suggests that MAG-induced axonal protection depends on the functional binding site around arginine 118.
The ectodomain R118 of MAG has been reported to be necessary for its lectin activity and may suggest a possible association of MAG with sialic-acid residues on the axonal surface for the protective effects. We observed that MAG did not promote axonal stability or prevent axonal degeneration in DRG cultures from B4galnt1 knockout mice lacking complex gangliosides (Fig. 4d). Although our data suggest that the axonal protective effect of MAG depends on the interaction with complex gangliosides, the nature of the interaction is beyond the scope of this study. Gangliosides could act as a co-receptor, binding receptor, or facilitator for clustering the full signaling complex assembly and cytoskeletal association within the membrane required for initiation of signal transduction.
New data in this report support the conclusion that MAG, a constituent of the adaxonal membrane of myelin-forming cells, promotes axonal stability and survival in cell culture and in vivo. While this effect on axonal stability is independent of Nogo signaling in the axon, it depends on arginine 118 in the RGD domain of the extracellular segment of the molecule. MAG signals to the axon to promote stability of axonal microtubules, and promotes axonal survival in the face of insults such as vincristine, acrylamide and inflammatory mediators. This protective effect may be augmented by effects on phosphorylation of MAP proteins and tau (Dashiell et al., 2002; Zheng et al., 2007). These may also affect axonal transport, which is known to be affected locally by demyelination (de Waegh et al., 1992). Moreover, MAG is known to locally increase axonal diameter (Yin et al., 1998; Dashiell et al., 2002). Its roles in limiting longitudinal growth is well-known, and its presence in the internodal and paranodal adaxonal plasmalemma of the myelin-forming cell means that it is likely to inhibit collateral sprouting along the course of the myelinated fiber. In these ways MAG discourages plasticity of the mature myelinated axon (Griffin and Thompson, 2008). These data, taken together with previous studies, suggest that the biological roles of MAG include promoting axonal stability, survival, and maximal radial caliber, and discouraging either longitudinal or collateral growth. It thereby encourages stable and rapid point-to-point impulse transmission (Griffin and Thompson, 2008).
We speculate that the loss of axonal stabilizing and protective effects of MAG may contribute to the late axonal degeneration seen in demyelinating diseases. Even though MAG is not a component of compact myelin, MAG is known to be lost from oligodendrocytes and Schwann cells that have undergone demyelination. There is a wealth of evidence that axons are liable to both prompt and progressive late Wallerian-like degeneration after demyelination (Dyck, 1975; Trapp et al., 1998; Scherer, 1999; Berciano et al., 2000; Krajewski et al., 2000; Bjartmar et al., 2003; Oh et al., 2004). In the PNS this is exemplified by demyelinating forms of Charcot-Marie-Tooth disease (CMT1), the most common forms of inherited peripheral neuropathy. In several forms of demyelinating forms of CMT, the genetic defects are in components of the Schwann cells, such as PMP-22 and P0 (Scherer, 1999). These defects lead to recurrent demyelination that can be detected by electrophysiologic changes early in childhood. However, the clinical manifestations often occur only years later and are due to progressive distal axonal loss in the peripheral nerves (Scherer, 1999). Distal axonal degeneration has been demonstrated in animal models of CMT, including P0, connexin 32, and PMP22 mutations in the PNS and PLP mutations in the CNS (Scherer, 1999). Importantly, axonal degeneration is now recognized to be a major component of the later progressive stage of the CNS inflammatory demyelinating disease, multiple sclerosis (MS). Undoubtedly axonal injury can be produced by inflammatory mediators, but it is likely that demyelination renders the axon more vulnerable to axonal degeneration (Bjartmar et al., 2003). The loss of the axonal stabilizing and protective effects of adaxonal MAG may contribute to this type of “secondary” axonal loss.
An active field at the moment is developing therapies to overcome the effects of MAG that block axonal regeneration in the CNS. Such therapies will need to avoid destabilizing injured and uninjured axons, thus rendering them more vulnerable to axonal degeneration.
We thank Douglas Kerr and Harish Pant for technical help. This work is supported by grants from the National Multiple Sclerosis Society Tissue Repair Grant (R 3760-A-3), the Nancy Davis Center Without Walls, and the National Institute of Health (K08 NS055135, R37 NS037096).
Competing Interest Statement The authors declare that they have no competing financial interests.