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Functional recovery after adult CNS damage is limited in part by myelin inhibitors of axonal regrowth. Three molecules, Nogo-A, MAG and OMgp, are produced by oligodendrocytes and share neuronal receptor mechanisms through NgR1 and PirB. While each has an axon inhibitory role in vitro, their in vivo interactions and relative potencies have not been defined. Here, we compared mice singly, doubly or triply mutant for these three myelin inhibitor proteins. The myelin extracted from Nogo-A mutant mice is less inhibitory for axons than is that from wild type mice, but myelin lacking MAG and OMgp is indistinguishable from control. However, myelin lacking all three inhibitors is less inhibitory than Nogo-A-deficient myelin, uncovering a redundant and synergistic role for all three proteins in axonal growth inhibition. Spinal cord injury studies revealed an identical in vivo hierarchy of these three myelin proteins. Loss of Nogo-A allows corticospinal and raphespinal axon growth above and below the injury, as well as greater behavioral recovery than in wild type or heterozygous mutant mice. In contrast, deletion of MAG and OMgp stimulates neither axonal growth nor enhanced locomotion. The triple mutant mice exhibit greater axonal growth and improved locomotion, consistent with a principal role for Nogo-A and synergistic actions for MAG and OMgp, presumably through shared receptors. These data support the hypothesis that targeting all three myelin ligands, as with NgR1 decoy receptor, provides the optimal chance for overcoming myelin inhibition and improving neurological function.
Neurological recovery after trauma to the adult mammalian central nervous system is limited by the inability of damaged axons to reconnect to their physiological targets. Axon regeneration is restricted by cell-autonomous factors (Bulsara et al., 2002; Szpara et al., 2007), by the astroglial scar (Yiu and He, 2006; Busch and Silver, 2007) and by CNS myelin (McGee and Strittmatter, 2003; Liu et al., 2006). Myelin-derived inhibitory proteins include NogoA (Chen et al., 2000; GrandPre et al., 2000; Prinjha et al., 2000), MAG (McKerracher et al., 1994; Mukhopadhyay et al., 1994), OMgp (Mikol and Stefansson, 1988; Wang et al., 2002a), RGM (Hata et al., 2006), ephrinB3 (Benson et al., 2005), semaphorins (Moreau-Fauvarque et al., 2003) and netrins (Low et al., 2008). Of these NogoA, MAG and OMgp have been studied most extensively. Though they do not share sequence homology they do share receptor signaling pathways. Neuronal NgR1 binds and transduces signals from NogoA, MAG and OMgp (Fournier et al., 2001; Domeniconi et al., 2002; Liu et al., 2002; Wang et al., 2002a; Barton et al., 2003; Hu et al., 2005; Venkatesh et al., 2005; Lauren et al., 2007), additionally NgR2 can also mediate MAG signaling (Venkatesh et al., 2005; Lauren et al., 2007). More recently, PirB has also been shown to bind and transduce signals from all three proteins in vitro (Atwal et al., 2008). Both NgR1 and PirB are implicated in adult brain plasticity separate from injury (McGee et al., 2005; Syken et al., 2006).
Though axon-inhibiting roles and receptor mechanisms for NogoA, MAG and OMgp have been demonstrated in tissue culture, their relative contribution to the inhibitory activity associated with CNS myelin in vivo remains undefined. In spinal cord injury studies, Nogo-A gene deficient mice are reported to have increased axonal growth of both sprouting and regenerative patterns (Kim et al., 2003; Simonen et al., 2003), however this phenotype varies with age (Kim et al., 2003; Simonen et al., 2003; Cafferty et al., 2007a), strain background (Dimou et al., 2006), genetic mutation (Zheng et al., 2003; Cafferty et al., 2007a; Steward et al., 2007) and type of lesion (Cafferty et al., 2007b). Mice deficient in OMgp display limited axon growth after SCI (Ji et al., 2008), and MAG deficient mice show no enhancement of axonal growth after SCI (Bartsch, 1996). Because these proteins have shared receptors and cellular mechanisms, they may be at least partially redundant in myelin for limiting axonal growth after injury.
To assess functional redundancy and axon-inhibitory hierarchy between ligands, we compared mice with single (nogoab−/−), double (mag−/−omgp−/−) or triple (nogoab−/−mag−/−omgp−/−) mutations in these myelin inhibitors. Analysis of myelin-induced inhibition of neurite outgrowth in vitro, axonal growth of corticospinal and raphespinal fibers in vivo and from behavioral studies produce similar findings. Nogo-A plays a demonstrable non-redundant role in limiting axonal growth, whereas MAG/OMgp do not. However, MAG/OMgp have a readily apparent axonal growth limiting effect on a Nogo-A deficient background consistent with redundant, synergistic axonal growth inhibition.
Nogo-abtrap/trap mice have been described (Kim et al., 2003; Cafferty et al., 2007a) and were backcrossed to C57BL/6 for 9 generations. This line contains a gene trap insertion in the largest Nogo-a selective exon and eliminates expression of Nogo-A and Nogo-B protein(Kim et al., 2003). The mag−/− mice were purchased from Jackson Labs identifier B6.Cg-Magtm1Rod/J (Bartsch et al., 1995) and were intercrossed with omgp−/− mice maintained on a C57BL/6 background (Ji et al., 2008), until homozygote double knockouts were produced. The omgp−/− mice were a generous gift of Dr. Sha Mi of BiogenIdec, Inc. The mag−/−omgp−/− mice were then intercrossed with nogoabtrap/trap mice to produce nogoabtrap/trapmag−/−omgp−/− mice. Single nogoabtrap/trap (N−/−), double mag−/−omgp−/− (MO−/−) and triple nogoabtrap/trapmag−/−omgp−/− (NMO−/−) mice were crossed with C57BL/6 to produce respective single, double and triple littermate heterozygotes.
Adult (2–4 months) female wild type (n = 10), nogoabtrap/trap (n = 12), nogoabwt/trap (n = 9), mag−/−omgp−/− (n = 12), mag+/−omgp+/− (n = 8), nogoabtrap/trapmag−/−omgp−/− (n = 48 dorsal hemisection, n = 8 sham lesioned), nogoabwt/trapmag+/−omgp+/− (n = 20 dorsal hemisection, n = 20 sham lesion) mice were deeply anesthetized with intraperitoneal ketamine (100mg/kg) and xylazine (15mg/kg). A laminectomy was performed to expose the dorsal portion of spinal cord corresponding to T6 and T7 levels. The dura mater was pierced and the spinal cord exposed, a pledget of gelfoam soaked in 1% lidocaine was placed on the exposed cord for 1 minute prior to lesion. A dorsal hemisection lesion was performed at T6 with a 30-guage needle and a pair of microscissors to a depth of 1.0 mm to completely sever the dorsal and dorsolateral corticospinal tract. In one cohort of animals the lesion depth was greater, 1.4 mm, producing a near total transection. For sham surgery, the laminectomy and exposure were identical but no spinal cord lesion was created. The overlying muscle and skin was sutured with 4.0 vicryl.
Four weeks after spinal cord injury, mice received unilateral cortical injections with Biotin Dextran Amine (BDA, 10,000 mol wt, Invitrogen) to antereogradely label the corticospinal tract as described (Kim et al., 2003; Kim et al., 2004; Cafferty et al., 2007b). Briefly, burr holes were made over the sensorimotor cortex and 4 microinjections were made to a depth of 1.0 mm (co-ordinates, 0.5–1.5 mm posterior to bregma and 0.5–1.5 mm lateral to bregma) to deliver a total volume of 1.2 μl of BDA. Six weeks after dorsal hemisection or sham lesion, mice were perfused with 4% paraformaldehyde. Post-fixed overnight at 4°C and embedded in 10% gelatin for immunohistochemical processing.
Four animals were removed from the study due to incomplete lesions as determined by >50% spared tissue by anti-GFAP staining (1 nogoabtrap/trap, 1 mag−/−omgp−/− and 2 nogoabtrap/trapmag−/−omgp−/− mice).
Mice that underwent dorsal hemisection lesions were assessed using the BMS scale (Basso et al., 2006). All measurements were collected by observers unaware of genotype. Data are presented as average ± SEM. Repeated Measures ANOVA with TUKEY post hoc analysis was completed to assess statistical differences between genotypes. Subsequent ANOVA analysis was completed to assess differences between genotypes at each time point post lesion.
Cortex was freshly dissected from wild type, nogoabtrap/trap, mag−/−omgp−/− and nogoabtrap/trapmag−/−omgp−/− mice and immediately homogenized in lysis buffer (10 mM Tris/HCl, 1% NP-40, 150 mM NaCl, 0.1% SDS, 1% deoxycholate, supplemented with protease inhibitors), sonicated and the supernatant was collected after centrifugation at 100,000 × g for 30 min. Protein (50 μg) was separated by SDS-PAGE and blotted onto PVDF. Membranes were probed with antibodies to detect Nogo-A (affinity purified Rabbit anti-Nogo-A (Wang et al., 2002b), MAG (1:1000, R&D Systems), OMgp (1:1000, R&D Systems), NgR1 (1:1000, R&D Systems), MBP (1:2500, Sigma) and GAPDH (1:25,000, Sigma). Immunoreactivity was visualized with anti-rabbit, anti-goat and anti-mouse IRDye 680 or 800 (1:10,000, Rockland) – conjugated secondary antibodies. Protein bands were detected with the Li-Cor Odyssey system (Li-Cor Biosciences).
Adult wild type dorsal root ganglia neurons were dissociated as previously described (GrandPre et al., 2000). DRG cells were cultured in Poly-D-Lysine pre-coated 96-well plates (Biocoat, Becton Dickinson Labware), which had been pre-incubated with myelin and laminin. Myelin was prepared (Norton and Poduslo, 1973; Robak et al., 2009) and extracted with 60 mM CHAPS (Spillmann et al., 1998) before dialysis against 10 mM HEPES, 150 mM NaCl, pH 7.4. 100 μl of 10 μg/ml extracted myelin from wild type, nogoabtrap/trap, mag−/−omgp−/− and nogoabtrap/trapmag−/−omgp−/− mice and vehicle (dialyzed extraction buffer alone) was incubated in each experimental well of a 96-well tissue culture plate for 2 hours at 37°C. Myelin extracts were then aspirated and immediately replaced with 100 μl of 10 μg/ml laminin (Sigma). After a further 2 hours of incubation at 37°C, laminin was aspirated. Wells were washed once with DMEM (Invitrogen) and immediately flooded with culture media (DMEM + B27, Invitrogen) containing dissociated DRG cells. Cells were cultured for 18 hours at 37°C and 5% CO2, after which time they were fixed by addition of an equal volume of 8% paraformaldehyde in 20% sucrose for 1 hour, and then washed with PBS. Cells were stained with antibodies to βIII-tubulin (1:5,000, Promega) and visualized with Alexa-Fluor 488 conjugated secondary antibodies. Analysis of neurite outgrowth was completed using ImageExpress automated imaging and neurite measurement algorithms without subjective input from the experimenter. Myelin was prepared independently from 4 mice of each genotype. Each independent myelin preparation was exposed to wild type DRG cells from 4 different mice with each preparation tested in quadruplicate on 4 separate days. Data are presented as mean neurite outgrowth ± SEM from 64 wells for each myelin genotype. In each well, more than 50 neurons were measured and average outgrowth per well calculated, so that in total more than 3000 neurons were included in each measurement.
Gelatin embedded brain and spinal cord were cut on a vibratome (Leica Microsystems, VT1000) and free floating sections were collected and stored in PBS/0.01% sodium azide. Sections (30 μm) of brain and spinal cord were immunostained with antibodies to detect myelin (1:200, Brain Stain Kit, Invitrogen), NeuN (1:1 000, Millipore), 5HT (1:5 000, Immunostar) and GFAP (1:10 000, DAKO) with appropriate Alexa Flour 488 or 568 (Invitrogen) conjugated secondary antibodies. Biotin Dextran Amine (BDA) detection was completed as previously described (Kim et al., 2003), using a nickel-enhanced diaminobenzidene (DAB) reaction. DAB and immuno-fluorescent images were collected on a Z1 Imager Zeiss microscope equipped with Apotome (Zeiss, Thornwood, NY).
All anatomical assessments were made by an observer unaware of the genotype.
For quantification of BDA+ CST axons crossing the midline, 5 randomly chosen transverse sections of cervical spinal cord were collected from each of 8–10 mice of each genotype and processed to visualize BDA+ CST axons with DAB. The average number of crossing fibers was recorded for each animal. Data represents average number of axons clearly crossing through lamina X ± SEM. Statistical significance was assessed using ANOVA with TUKEY post hoc analysis.
For quantification of the number of CST fibers growing up to and past the lesion site, half of all the sagittal sections from each mouse (~20 sections) were processed to visualize BDA+ CST axons with DAB. The sum of axons crossing perpendicular lines at 3 and 1 mm rostral to the lesion and 0, 1, 2 and 3 mm caudal to the lesion was recorded in all 20 sections from each animal. Data represent the average raw number of axons ± SEM. Statistical significance was assessed using ANOVA with TUKEY post hoc analysis.
The density of 5HT-IR raphespinal innervation of ventral horn was determined using NIH Image J version 1.62. Labeled fibers were selected by thresholding, and average fiber length within gray matter was measured after utilizing the skeletonize function in 5 sections from each mouse. Data represents average 5HT density ± SEM/genotype. Statistical significance was assessed using ANOVA with TUKEY post hoc analysis.
Lesion depth was assessed in 5 sagittal sections from each lesioned mouse using GFAP immunoreactivity to delineate the lesion margins. We measured the dorso-ventral extent of hyperfilamentous GFAP-IR for each animal. Animals that illustrated less then 50% hyperfilamentous GFAP-IR in the dorso-ventral axis were removed from the study (one NMO−/− mouse and two NMO+/− mice). Data represents average GFAP-IR depth ± SEM/genotype. Statistical significance was assessed using ANOVA with TUKEY post hoc analysis.
In order to ascertain the interactions and the relative potencies of Nogo, MAG and OMgp to inhibit axon growth, we generated single, double and triple null mutant mice for Nogo (N−/−), MAG/OMgp (MO−/−) and Nogo/MAG/OMgp (NMO−/−) respectively. The mag−/−mice (Bartsch et al., 1995) were bred with omgp−/− mice (Ji et al., 2008) and backcrossed until homozygote double mutants were generated. Progeny from mag/omgp double knockouts were intercrossed with the nogoabtrap/trap mice (Kim et al., 2003; Cafferty et al., 2007a) to produce nogotrap/trapmag−/−omgp−/− triple knockout mice. Western blot analysis of cortical brain lysate confirms the absence of Nogo-A in single mutant mice, of MAG and OMgp in double mutant mice and of all three ligands in the triple mutant mice (Fig. 1 A). Expression of myelin basic protein (MBP) and NgR1 is similar in the mutant mice in comparison to wild type mice.
Discrete myelin abnormalities at particular ages have been reported for each of these three mutant strains (Li et al., 1994; Montag et al., 1994; Bartsch, 1996; Huang et al., 2005; Pernet et al., 2008), so we considered whether the double or triple mutant mice might display more pronounced abnormalities in overall CNS myelination. Regional patterns of myelination assessed by anti-MBP staining in brain and spinal cord of nogoabtrap/trap (Fig. 1 G, E), mag−/−omgp−/− (Fig. 1 L, O) and nogoabtrap/trapmag−/−omgp−/− mice (Fig. 1 Q, T), are identical to that observed in wild type mice (Fig. 1 B, C). Furthermore, there is no qualitative difference in cortical or medullary spinal cord neuronal number or distribution in nogoabtrap/trap (Fig. 1 H, I, K), mag−/−omgp−/− (Fig. 1 M, N, P) and nogoabtrap/trapmag−/−omgp−/− mice (Fig. 1 R, S, U). Thus, neuronal development is remarkably normal in mice lacking all three myelin inhibitors.
CNS myelin inhibition of neurite outgrowth from CNS or PNS neurons is readily detected in tissue culture (Schwab and Thoenen, 1985; Savio and Schwab, 1989). To investigate the relative roles of Nogo, MAG and OMgp in this inhibition, we prepared myelin extracts from nogoabtrap/trap, mag−/−omgp−/− and nogoabtrap/trapmag−/−omgp−/−. Dissociated dorsal root ganglia (DRG) neurons from adult wild type mice were cultured for 18 hours on 96-well tissue culture plates upon which 1 μg of extracted myelin from wild type mice, nogoabtrap/trap mice, mag−/−omgp−/− mice, nogoabtrap/trapmag−/−omgp−/− mice and dialyzed extraction buffer (vehicle) had been adsorbed without dehydration. DRG cells grown on plates coated with vehicle extend neurites with an average total length of 192.5 ± 11.6 μm (Fig. 1A, F), while cells grown on wild type myelin exhibit significantly reduced outgrowth with average total neurite length per well of 76.5 ± 13.5 μm (Fig. 2B, F, *p < 0.01, ANOVA). Myelin prepared from mag−/−omgp−/− mice (Fig. 2C, F) exhibit a non-significant trend to less inhibition than wild type myelin, suggesting that these proteins may have little role in myelin inhibition. In contrast, myelin prepared from nogoabtrap/trap mice is significantly less inhibitory for DRG neurite outgrowth in comparison to wild type (132.9 ± 1.8 μm vs. 76.5 ± 13.5 μm, **p < 0.01 ANOVA) and mag−/−omgp−/− myelin (132.9 ± 11.8 μm vs. 99 ± 10 μm, **p < 0.01 ANOVA Fig. 2D, F), demonstrating an independent and predominant role for Nogo in myelin inhibition.
Because Nogo, MAG and OMgp each interact with both NgR1 and PirB axonal receptors, we considered whether MAG and OMgp action might be uncovered in the absence of Nogo-A. Myelin prepared from nogoabtrap/trapmag−/−omgp−/− mice is significantly less inhibitory to neurite outgrowth (Fig. 2E, F) than wild type myelin (175.1 ± 25 μm vs. 76.5 ± 13.5 μm, # p < 0.01 ANOVA), mag−/−omgp−/− myelin (175.1 ± 25 μm vs. 99 ± 10 μm, # p < 0.01 ANOVA) or nogoabtrap/trap myelin (175.1 ± 25 μm vs. 132.9 ± 12.8 μm, # p < 0.01 ANOVA). Thus, the triple mutant preparation reveals a synergistic or ancillary role for MAG and OMgp in myelin limitation of axonal outgrowth on the Nogo-null background.
We sought to determine if a similar relationship between Nogo/MAG/OMgp genotype and axonal growth exists in vivo after traumatic injury. Dorsal hemisection (DhX) of the thoracic spinal cord results in interruption of many descending motor and ascending sensory spinal tracts, such that lesioned mice lose function in their hind limbs. Restoration of hind limb function depends largely on the re-establishment of axonal communication across the spinal cord injury site, either directly via long distance growth of injured fibers around or through the lesion site, or indirectly through local growth of injured and intact fibers to create new bypass circuits. We assessed the contribution of Nogo, MAG and OMgp to these various patterns of axon growth and recovery.
As a first step towards evaluating these multiple mechanisms, we measured the local growth of CST axons within the cervical enlargement at levels C5–C8, several mm rostral the lesion in mice of multiple genotypes. We labeled the CST via BDA microinjection into the right motor cortex (Supplementary Figure S1). Uninjured intact wild type (Fig. 3A, B), nogoabtrap/trap (Fig. 3E, F), mag−/−omgp−/− (Fig. 3C, D) and nogoabtrap/trapmag−/−omgp−/− mice (Fig. 3G, H) displayed normal CST fasciculation in the ventral dorsal columns, termination of fibers in grey matter and the sparse presence of fibers in the dorsolateral funiculi (Fig. 3A–H) in the cervical cord. There was no significant difference in the number of BDA+ fibers observed crossing the midline and entering cervical spinal grey matter on the ipsilateral side of sham lesioned wild type, nogoabtrap/trap, mag−/−omgp−/− and nogoabtrap/trapmag−/−omgp−/− mice (Fig. 3Q, black bars). Six weeks after DhX, a small number of lesioned BDA+ fibers are seen crossing the midline in wild type (Fig. 3I, J) and mag−/−omgp−/− (Fig. 3K, L) mice, however the number differs insignificantly from sham lesioned littermates (Fig. 3Q). Thus, injury may produce minor degrees of CST sprouting but the absence of MAG and OMgp does not alter this growth, paralleling the neurite outgrowth assays in the presence of myelin (Fig. 2).
In contrast to control mice, nogoabtrap/trap mice exhibit significantly increased numbers of BDA+ CST fibers crossing the midline after DhX in comparison to intact and lesioned wild type or mag−/−omgp−/− mice, or intact nogoabtrap/trap mice (Fig. 3M, N, Q, * p < 0.001, # p < 0.01, ANOVA). Even greater degrees of BDA+ CST sprouting across the midline is observed in nogoabtrap/trapmag−/−omgp−/− mice after DhX in comparison to intact and lesioned wild type, mag−/−omgp−/−, nogoabtrap/trap mice and intact nogoabtrap/trapmag−/−omgp−/− mice (Fig. 3 O, P, Q, ** p < 0.001, ## p < 0.01, ANOVA). The reproducibility of this phenotype is illustrated in Suppl. Fig. S2. Thus, the absence of MAG/OMgp increases a Nogo-deficient post-injury axonal sprouting phenotype, and this matches the in vitro myelin inhibition of axonal growth (Fig. 2).
The sprouting of CST fibers in the cervical spinal cord of nogoabtrap/trap and nogoabtrap/trapmag−/−omgp−/− mice rostral to the T8 dorsal hemisection indicates that intact spinal grey and white matter are less inhibitory to axon growth. In order to test whether lesioned areas are more permissive for axon growth in nogoabtrap/trap, mag−/−omgp−/− and nogoabtrap/trapmag−/−omgp−/− mice in comparison to wild type mice, we assessed the growth of BDA+ CST fibers in sagittal sections spanning a T8 dorsal hemisection (Fig. 4A–P). The CST fails to grow caudal to the DhX in wild type mice (Fig. 4A). CST axons are seen at the lesion perimeter, and most of these axons exhibit dystrophic end bulbs characteristic of abortive regeneration (Fig. 4 Aii, arrows, (Silver and Miller, 2004)). Camera Lucida reconstruction of serial sagittal sections through the lesion site illustrates the absence of BDA+ CST axons caudal to the lesion (Fig. 4B) in wild type mice after DhX, the same phenotype was observed in nogoabWT/trapmag+/−omgp+/− mice (Fig. 4C–D) and mag−/−omgp−/−mice (Fig. 4 G–H). Significant numbers of regenerating BDA+ CST fibers were observed growing at 1, 2, and 3 mm past the lesion site in nogoabtrap/trap (Fig. 4 E, F, Q, * p < 0.01 ANOVA) and nogoabtrap/trapmag−/−omgp−/− mice (Fig. 4I–P, Q, ** p < 0.01 ANOVA) in comparison to wild type and nogoabWT/trapmag+/−omgp+/− mice. Furthermore, nogoabtrap/trapmag−/−omgp−/− mice displayed significantly more BDA+ CST axons after DhX at 1, 2 and 3 mm caudal to the lesion site in comparison to nogoabtrap/trap mice (Fig. 4Q, ** p < 0.001 ANOVA). High power photomicrographs i and ii illustrate the irregular growth patterns, characteristic (Steward et al., 2003) of regenerated fibers. The pattern of increased growth in the absence of Nogo increased by the absence of MAG and OMgp parallels the in vitro findings and rostral CST sprouting results.
We sought to ascertain whether regenerating fibers crossing the lesion in nogoabtrap/trapmag−/−omgp−/− mice preferentially grew in white or grey matter. We collected serial sections from a nogoabtrap/trapmag−/−omgp−/− mouse and reconstructed one side of the spinal cord using all sections with evidence of BDA+ axons (Fig. 5A–J). Photomicrographs A, C, E, G are projection images of 4 sagittal sections and represent 4 regions of spinal cord depicted in schematic. The green represents for the central zone (mostly dorsal and ventral column white matter and central canal), blue for the medial zone (mostly dorsal and ventral grey matter), yellow for the medio-lateral zone (grey > white matter) and red for the lateral zone (grey < white mater). Camera Lucida reconstructions B, D, F and H represent each of the collapsed images in the projection in a distinct color. Most axons caudal to the lesion site appear in zones with a greater percentage of white matter. The percentage of BDA+ CST axons observed in white matter 2 mm caudal to the lesion site in nogoabtrap/trapmag−/−omgp−/− mice is 62.1 ± 1.4 % (n = 9, ± SEM).
To confirm that the large number of axons present at and past the lesion site (Fig. 5K–L) reflect regenerative growth, we immunostained sagittal sections of spinal cord from each of the 4 zones with GFAP to observe the extent of injury-induced astrocyte activation (see below). The GFAP-IR images were projected onto the Camera Lucida reconstruction of axonal growth (Fig. 5I) to demarcate the lesion site relative to regenerating axons (Fig. 5J). GFAP-IR can be seen extending from the dorsal surface of the spinal cord to below the central canal (Fig. 5J, O). Thus, reconstruction demonstrates that CST fibers regenerate extensively into white matter greater than gray matter in the caudal spinal cord of nogoabtrap/trapmag−/−omgp−/− mice.
The DhX injury damages multiple spinal tracts in addition to the CST, including the serotonergic raphepsinal tract (RST). The RST contributes significantly to locomotion (Lemon, 2008). To determine whether the growth of raphespinal axons is differentially sensitive to the presence of NogoA/B, MAG and OMgp, we assessed the growth pattern of the RST after DhX in wild type, nogoabtrap/trap, mag−/−omgp−/− and nogoabtrap/trapmag−/−omgp−/−mice. 5HT-immunoreactive (5HT-IR) RST axons originating in the brainstem descend bilaterally in the lateral columns and densely innervate both dorsal and ventral grey matter at all spinal levels. We focused our analysis on the ventral horn of the lumbar spinal cord where the RST is know to synapse on motorneurons (Mason, 2001). 5HT-IR axons are observed to ramify densely in the L4/5 ventral horn of sham lesioned wild type and nogoabtrap/trapmag−/−omgp−/− mice (Fig. 6A, B). DhX at T8 significantly reduces the density of 5HT-IR by 75% in wild type (Fig. 6 C, D, K, * p < 0.001 ANOVA) and mag−/−omgp−/− mice (Fig. 6E, F, K, * p < 0.001 ANOVA). The reduction of caudal 5HT fibers is less pronounced in nogoabtrap/trap mice (Fig. 6G, H, K, * p < 0.001 ANOVA). In obvious distinction, the density of 5HT innervation in the L4/5 ventral horn of nogoabtrap/trapmag−/−omgp−/− mice is restored fully, being insignificantly different from sham lesioned wild type or nogoabtrap/trapmag−/−omgp−/− control mice (Fig. 6I, J, K). The intact RST innervation likely reflects caudal sprouting of these fibers after the injury to restore normal fiber density. As for other measures, there is no regenerative phenotype in mag−/−omgp−/− mice, but the partial phenotype in the nogoabtrap/trap mice is enhanced by deletion of MAG and OMgp.
The primary outcome in this study is the degree of axonal growth after spinal lesion in mice of different genotypes. However, we also scored functional outcomes by observing locomotion in the open field for each cohort. Standardizing lesion severity in experimental SCI models is crucial to correlating anatomical with functional outcomes. Therefore, we assessed lesion depth in animals that underwent dorsal hemisection (Fig. 7A–D), using GFAP immunoreactvity. Wild type, nogoabtrap/trap (Fig. 7A), mag−/−omgp−/− (Fig. 7B), nogoabWT/trapmag+/−omgp+/− (Fig. 7C) and nogoabWT/trapmag+/−omgp+/− nogoabtrap/trapmag−/−omgp−/− mice (Fig. 7D) display equivalent induction of GFAP at the lesion site 5 weeks post lesion. The percentage of GFAP-negative spared spinal tissue in the remaining groups of animals is insignificantly different between genotypes (Fig. 7E).
Mice behavior was assessed on day 3 pre-lesion, and 6 hours, 3, 7, 14, 21 and 28-days post lesion (Fig. 7F–J). All animals display flaccid hind limb paralysis 6 hours after lesion and slowly regain function over the observation period. Only nogoabtrap/trap and nogoabtrap/trapmag−/−omgp−/− mice recovered significant hind limb function in comparison to nogoabwt/trap (Fig. 7F, Repeated Measures ANOVA p < 0.001) and nogoabwt/trapmag+/−omgp+/− control mice (Fig. 7H, Repeated Measures ANOVA p < 0.001), mag−/−omgp−/− mice failed to recover significant function in comparison to mag+/−omgp+/− mice (Fig. 7 G). There was no significant difference in BMS scores between wild type, nogoabwt/trap, mag+/− omgp+/− and nogoabwt/trapmag+/−omgp+/− control mice after DhX (Fig. 7I). Therefore we pooled the control groups (Fig. 7J, WT/HT) for comparison to single, double and triple mutants. Both nogoabtrap/trap and nogoabtrap/trapmag−/−omgp−/− mice recovered significant function in comparison to controls (Fig. 7J, Repeated Measures ANOVA, p < 0.005 and p < 0.001 respectively). Furthermore nogoabtrap/trapmag−/−omgp−/− mice recovered to a greater degree than do nogoabtrap/trap mice (Repeated Measures ANOVA, p < 0.05). There was no significant difference between mag−/−omgp−/− and control mice.
To support the robustness of this finding across injury severity, we examined a separate cohort of nogoabtrap/trapmag−/−omgp−/− and nogoabWT/trapmag+/−omgp+/− that had a more severe, near total transection lesion due to deeper cutting at the time of surgery. Such mice preserve less than 10% of tissue (Fig. 8A–D). The severely injured nogoabtrap/trapmag−/−omgp−/− mice recover greater function in the open field than do the nogoabWT/trapmag+/−omgp+/− mice (Fig. 8E, Repeated Measures ANOVA p < 0.001).
The major conclusions from this work are that Nogo-A is the myelin inhibitor with the greatest inhibitory activity, and that MAG and OMgp play synergistic roles in preventing axonal growth in the adult mammalian CNS. These conclusions are supported by analysis of axonal outgrowth in vitro on substrates coated with myelin prepared form mice of various mutant genotypes. The MAG/OMgp mutant myelin is as inhibitory as wild type myelin. The reduced inhibition generated by deletion of Nogo-A is enhanced by deletion of MAG/OMgp as well. A similar synergy of MAG plus OMgp with Nogo-A is observed in spinal cord injured mice through examination of rostral CST sprouting, CST regeneration across the lesion, caudal RST sprouting and locomotor recovery. We conclude that Nogo-A is a principal myelin inhibitor, with a redundant and synergistic role for MAG plus OMgp.
The myelin extracted from mag−/−omgp−/− mice is as inhibitory as wild type myelin in neurite outgrowth from wild type DRG neurons. We chose to use sensory neurons as they express NgR1 (Fournier et al., 2001), NgR2 (Venkatesh et al., 2005), Lingo-1 (Mi et al., 2004), p75 (McMahon et al., 1994), TAJ/TROY (Park et al., 2005; Shao et al., 2005) and PirB (Atwal et al., 2008); all the receptor components that have been reported necessary to transduce Nogo-A, MAG and OMgp binding. The lack of significant dis-inhibition observed on myelin from mag−/−omgp−/− mice may be due to our extraction protocol, which may have failed to collect the axogliasome OMgp-rich fraction (Huang et al., 2005). However, previous studies have also reported a lack of enhanced neurite outgrowth of DRG cells grown on 1 μg of extracted myelin from mag−/− (Bartsch et al., 1995; Ng et al., 1996) and omgp−/− mice (Ji et al., 2008). Both of these studies concluded that other inhibitors present in CNS myelin mask the potential benefit that removing either one of these two proteins would otherwise show. Accordingly, we compared the outgrowth inhibitory activity of myelin from nogoabtrap/trap and nogoabtrap/trapmag−/−omgp−/− mice. As previously reported (Kim et al., 2003; Simonen et al., 2003; Zheng et al., 2003), we find that myelin extracted from nogoab−/− mice was less inhibitory to neurite outgrowth in comparison to myelin from wild type mice. Critically, we find that nogoabtrap/trapmag−/−omgp−/−myelin is less inhibitory than nogoabtrap/trap myelin. In fact, there is no detectable inhibition of axonal growth by nogoabtrap/trapmag−/−omgp−/− myelin. It should be noted that we used a detergent extraction protocol for isolating myelin. This may explain why there is no remaining inhibition from other proteins reported to be associated with crude myelin, such as ephrinB3 (Benson et al., 2005) and netrin-1 (Low et al., 2008). Under these assay conditions, Nogo-A, MAG and OMgp fully account for CNS myelin inhibition of axonal outgrowth.
In accord with previous reports for single mag−/− (Bartsch et al., 1995) and omgp−/− (Ji et al., 2008) mice, we fail to observe growth of CST axons above the lesion, or of CST plus RST axons below the lesion after dorsal hemisection in wild type or mag−/−omgp−/− mice. In line with our previous data, we find that nogoabtrap/trap mice display a significantly greater number of CST axons sprouting above the lesion and regenerating past the lesion (Kim et al., 2003; Cafferty et al., 2007a), as well as enhanced RST innervation of the lumbar ventral horn. Most importantly, these phenotypes are augmented in nogoabtrap/trapmag−/−omgp−/− mice. Thus, the in vivo anatomical outcome from SCI exactly parallels the in vitro axonal outgrowth results with regard to the relative roles of Nogo-A, MAG and OMgp.
Although the primary focus of these studies was axonal outgrowth inhibition by myelin, but we did monitor BMS scores as a neurological outcome after SCI. As in the axonal assays, the mag−/−omgp−/− mice behave indistinguishably from wild type or heterozygous controls. Despite this lack of direct effect, MAG and OMgp expression play a role in the recovery in mice lacking Nogo-A expression. Recovery is significantly greater when all three NgR1/PirB ligands are absent. The recovery of the triple mutant mice is dramatically improved compared to control mice, reaching a BMS score of 5.9 ± 0.4 vs 3.4 ± 0.2.
Striking axonal growth in nogoabtrap/trapmag−/−omgp−/− mice includes the sprouting of rostral CST fibers and caudal RST fibers. These zones are largely intact and devoid of gliotic reaction. The sprouting phenotypes are similar to the pyramidotomy-induced CST sprouting observed in ngr1−/− and nogoaatg/atg mice (Cafferty and Strittmatter, 2006). The zone of overt of glial scarring at the lesion site shows regenerative growth, although this is less prolific than the axonal sprouting far from the lesion. The greater degree of plasticity-linked sprouting than frank regeneration is consistent with the deletion of myelin inhibitors not altering the inhibitory extracellular matrix enriched at the lesion site (for review see, (Busch and Silver, 2007). Furthermore, many other inhibitory proteins that were not targeted in this study remain in nogoabtrap/trapmag−/−omgp−/− mice include ephrinB3 (Benson et al., 2005), netrin-1 (Low et al., 2008), SEMA6A (Runker et al., 2008), SEMA4D (Moreau-Fauvarque et al., 2003) and RGMa (Hata et al., 2006).
Previous evaluation of mag−/− and omgp−/− mice has identified important roles for these proteins separate from function as inhibitors of neurite outgrowth. MAG is crucial for the initiation and maintenance of myelination in the CNS. Consistent with MAG localization to paranodal regions, ultrastructural analysis of mag−/− mice demonstrates redundant myelin loops, supernumerary myelin sheaths and disorganization of the axo-glial junctions in the paranodal regions (for review see (Bartsch, 1996). Signaling downstream of MAG in oligodendrocytes may require specific splice forms of the MAG cytoplasmic tail and Fyn activation (Fujita et al., 1998; Biffiger et al., 2000).
OMgp was originally identified as being enriched in CNS white matter (Vourc’h and Andres, 2004), and is expressed by both oligodendrocytes and neurons (Huang et al., 2005). OMgp has been shown to have an anti-proliferative effect when over expressed in NIH3T3 cells (Habib et al., 1998) via PDGF dependent mechanism. PDGF is a known oligodendrocyte precursor cell (OPC) mitogen (Wolswijk et al., 1991), hence OMgp could determine OPC proliferation and subsequent differentiation. A recent study has also implicated OMgp in maintaining the stability of node of Ranvier, where it additionally may restrict aberrant axonal sprouting (Huang et al., 2005).
Nogo-A has also been reported to play a role in the developmental timing of myelination (Pernet et al., 2008). Despite these varied myelin phenotypes for Nogo, MAG and OMgp, there is no overt deficit at the light microscopy level in myelination or oligodendrocytes in adult triple mutant mice. Thus, these roles in myelin formation are unlikely to occur via shared synergistic mechanisms. Consistent with independent roles for these three proteins in myelination, no myelin phenotype has been reported for NgR1 null mice.
In light of the data presented here and the mild axon regenerative phenotype observed in both mag−/− and omgp−/− mice, it is clear that NogoA is the most potent NgR1/PirB ligand expressed in the adult spinal cord. Indeed targeting NogoA pharmacologically (Schnell and Schwab, 1990, 1993; Bregman et al., 1995; Brosamle et al., 2000; Liebscher et al., 2005) has been shown to enhance axonal regeneration and functional recovery after SCI in rodents. Genetic evidence to support the fact that NogoA is a key inhibitor of axonal regeneration has been less consistent, some studies report significant axonal growth after SCI in nogo−/− mice (Kim et al., 2003; Simonen et al., 2003; Dimou et al., 2006) while others fail to observe this phenotype (Zheng et al., 2003; Lee et al., 2009). While there may be uncertainty regarding the variables influencing the phenotypes of various nogo−/− mice, such as the age of lesioned mice, background strain of mice and nature of the mutant allele (Cafferty and Strittmatter, 2006; Dimou et al., 2006; Cafferty et al., 2007a; Steward et al., 2007), the efficacy of anti-NogoA therapies has been reported in non-human primate SCI models (Freund et al., 2006; Freund et al., 2007; Beaud et al., 2008; Freund et al., 2009) and is being tested now in the clinic.
While the current studies do not support separate targeting of either MAG or OMgp as a means to promote neurological recovery, they do demonstrate a clear benefit in all assays for targeting the three myelin ligands as a group rather than simply targeting Nogo-A alone. The molecular basis for this synergy is likely the shared receptors, NgR1 and PirB. The only published method for targeting all three ligands is the NgR1 decoy receptor (Fournier et al., 2002; Li et al., 2004; Li et al., 2005). Of note, the degree of CST axonal growth and neurological benefit observed after NgR(310)ecto-Fc treatment of dorsal hemisected rats was greater than with anti-Nogo antibody treatment (Li et al., 2004; Liebscher et al., 2005). Although these two studies were conducted in different laboratories, the results parallel those observed in the Nogo-A, MAG, OMgp triple mutant as compared to the Nogo-A single mutant mouse studies here. The greater benefit of targeting all three ligands may also explain the benefit of NgR(310)ecto-Fc treatment for spinal contusion recovery (Wang et al., 2006) in the absence of any report of benefit for anti-Nogo treatment of spinal contusion injury.
We conclude that combined targeting of NgR1/PirB ligands promotes axonal regeneration and functional recovery to a certain point. Surmounting this threshold will require targeting additional inhibitory proteins present in CNS, while concomitantly addressing the low intrinsic growth capacity of CNS neurons.
We thank Sha Mi of BiogenIdec for providing OMgp null mice, and Stefano Sodi for expert animal husbandry. This work is supported by research grants from the N.I.H. to W.B.J.C and to S.M.S. and by a grant from the Falk Medical Research Trust to S.M.S.