|Home | About | Journals | Submit | Contact Us | Français|
Glycosaminoglycan (GAG) side chains endow extracellular matrix proteoglycans with diversity and complexity based upon the length, composition, and charge distribution of the polysaccharide chain. Using cultured primary neurons, we show that specific sulfation in the GAG chains of chondroitin sulfate (CS) mediates neuronal guidance cues and axonal growth inhibition. Chondroitin-4-sulfate (CS-A), but not chondroitin-6-sulfate (CS-C), exhibits a strong negative guidance cue to mouse cerebellar granule neurons. Enzymatic and gene-based manipulations of 4-sulfation in the GAG side chains alter their ability to direct growing axons. Furthermore, 4-sulfated CS GAG chains are rapidly and significantly increased in regions that do not support axonal regeneration proximal to spinal cord lesions in mice. Thus, our findings provide the evidence showing that specific sulfation along the carbohydrate backbone carries instructions to regulate neuronal function.
Glycosaminoglycans are a widely distributed, structurally diverse family of sulfated, unbranched polysaccharides expressed abundantly on the surface of cells and incorporated into extracellular matrix (ECM) (Bishop et al., 2007). GAGs have emerged as important regulators of signaling involved in cell growth, tumorigenesis, and inflammation (Iozzo, 2005; Parish, 2006; Taylor and Gallo, 2006). One species of GAG that is uniquely important in morphogenesis, cell division, and cartilage development is chondroitin sulfate, the carbohydrate component of chondroitin sulfate proteoglycans (CSPGs), molecules that are spatiotemporally regulated during brain development (Hwang et al., 2003; Knudson and Knudson, 2001; Laabs et al., 2005; Sirko et al., 2007) and upregulated after injury in the central nervous system (CNS) (Chung et al., 2000; Silver and Miller, 2004).
During development, several different CSPGs have been localized to specific regions, such as the optic chiasm, where they appear to provide chemorepulsive signals to guide axonal growth (Bandtlow and Zimmermann, 2000; Chung et al., 2000; Ichijo and Kawabata, 2001). In the adult nervous system, high levels of CSPGs are found in perineuronal nets, where they are thought to stabilize synaptic connections. Removal of CS GAG chains with chondroitinase ABC (cABC) restores ocular dominance plasticity in the adult visual cortex of rats (Pizzorusso et al., 2002). Even higher levels of CSPGs are found after injuries to the adult mammalian CNS, where CSPGs are a major component of the glial scar that impedes axonal regeneration (Silver and Miller, 2004). cABC treatment enhances axonal growth and functional recovery after spinal cord injury (Bradbury et al., 2002). However, the distinctive features of CS GAG chains involved in these processes have not been fully identified.
CS GAG chains are complex unbranched polysaccharides of variable length with a backbone structure composed of a repeating disaccharide unit consisting of D-glucuronic acid (GlcA) and N-acetyl-D-galactosamine (GalNAc). This simple repetitive structure then can undergo extensive modification by sulfation at the C2 position of GlcA and/or the C4 or C6 position of GalNAc residues during biosynthesis (Reviewer’s Figure). The location of modifications by distinct sulfotransferases is not template-driven, leading to a huge number of potential combinations of sulfation along the carbohydrate backbone. Whether specific sulfation in chondroitin sulfate regulates biological events is a matter of conjecture.
In this study, we use axonal guidance/growth and specific modifications of the sulfation of CS GAG chains as a model to decipher the nature and importance of specific sulfation and the mechanisms by which it coordinates biological events. We present evidence that small changes in 4-sulfation of CS GAG chains have major effects on the potency of CSPGs to impart guidance cues to neurons. These results support the concept that distinct sulfation along the carbohydrate backbone carries instructions to regulate neuronal function.
We performed axonal guidance spot assays (Meiners et al., 1999) to determine the behavior of axons as they encounter immobilized CSPGs. Axonal behavior of cultured mouse cerebellar granule neurons (CGNs) was analyzed near a defined region of chicken CSPG immobilized onto poly-L-lysine (PLL)-coated coverslips. As observed previously (Laabs et al., 2007), most axons were deflected and few crossed onto the CSPG-rich area of the coverslip (Fig. 1A). Time-lapse imaging with adult mouse dorsal root ganglion neurons showed that filopodia dynamically sampled the CSPG spot (red), and that the growing axons turned at the interface between PLL and CSPG, and continued to extend along the interface, which is in contrast to growth cone collapse (Supplemental movie). Removal of the CS GAG chains by cABC abolished this negative axonal guidance cue, indicating that the repellant activity of CSPGs is specifically mediated by the CS GAG chains (Fig. 1B).
We examined whether CS GAG chains alone could repel axons. When 4-sulfate-enriched (84%, determined by HPLC, supplemental Fig. S1) CS-A was spotted onto PLL, axons were repelled at the interface in a manner comparable to native CSPGs (Fig. 1C and G). This repellent activity of CS-A was found to be very sensitive to cABC treatment. CS-A partially digested with cABC was precipitated with ethanol to remove disaccharides from the fractions, immobilized as spots, and subjected to axonal guidance spot assays. Digestion by the enzyme of less than 2% of the total GAG abolished its activity (Fig. 1D). This indicates that a small portion of CS GAG chains is essential for neuronal guidance activity. More surprising is that 6-sulfate-enriched (84%) CS-C had no inhibitory activity, as axons and cell bodies grew well on immobilized CS-C (Fig. 1E). These results suggest that sulfation at the C4 position of the GalNAc moiety presents a specific negative guidance cue to axons. Axons of dissociated embryonic mouse cortical neurons showed the same behavior to immobilized CS-A and CS-C (Supplemental Fig. S2).
The role of CS 4-sulfation in axonal guidance was further confirmed by the observation that chondro-4-sulfatase treatment of CS-A totally abolished the axon-repellant action (Fig. 1F and G), despite only a modest reduction in 4-sulfation (Table). This result strengthens the idea that subsets of the sulfation are crucial for its biological activity. To exclude the possibility that the presence of cABC in the chondro-4-sulfatase preparation was responsible for the drastic change in its biological activity, we conducted axonal guidance spot assays with CSPGs after treatment with chondro-4-sulfatase. Note that CS disaccharides are good substrates for chondro-4-sulfatase, but intact CSPGs are not (Yamagata et al., 1968). Axons were repelled by sulfatase-treated CSPGs, comparable to non-treated CSPGs (data not shown). Further, CS-A was extensively digested with chondro-4-sulfatase at 37°C for 16 hours and subjected to fluorescent labeling without cABC treatment. Since the appearance of CS disaccharides is dependent upon cABC activity, the presence or absence of fluorescent signals derived from CS disaccharides allows us to determine if cABC is a contaminant of the chondro-4-sulfatase. While we observed clear fluorescent signals in HPLC analysis with cABC treatment (Table), there was no signal derived from CS disaccharides when cABC treatment was skipped after chondro-4-sulfatase digestion (data not shown). Both of these experiments strongly suggest that it is the chondro-4-sulfatase that alters the biological activity of CS-A.
To explore the functional consequences of sulfation of CS GAG chains on axonal behavior, we examined neuron-astrocyte interactions using a co-culture system, which is more physiologically relevant. In the adult CNS, astrocytes are generally supportive of neuronal function. However, injuries to the CNS induce a gliotic reaction characterized by the presence of reactive astrocytes, which are major components of the glial scar considered to be detrimental to axonal regeneration. TGFβ is rapidly upregulated after CNS injury in vivo and is important both as a soluble regulator of ECM formation and in inducing reactive astrocytes (Flanders et al., 1998; Smith and Strunz, 2005). Confluent cultures of astrocytes were pretreated with TGFβ1 for 7 days; dissociated CGNs were plated onto these monolayers and co-cultured in fresh media without TGFβ1 for 2 days, followed by measurement of axonal length. Whereas axons of CGNs growing on untreated astrocytes elaborated long and thin processes (Fig. 2A, 93 ± 4 µm, mean ± SD process length), the axons of neurons cultured on TGFβ1-treated astrocytes were significantly shorter processes (54 ± 2 µm, P < 0.01 compared to untreated astrocytes, Student’s t-test). This reduction in axonal growth was also observed when neurons alone were cultured in conditioned media (CM) derived from TGFβ1-treated astrocytes (Fig. 2B). To exclude the possibility that TGFβ1 directly affects axonal growth, a potent TGFβ type I receptor inhibitor, SB-431542, was added to CM derived from TGFβ1-treated astrocytes. SB-431542 addition failed to restore neuronal growth, confirming that TGFβ1-dependent axonal growth inhibition is mediated through its action on astrocytes and not neurons.
Consistent with axonal growth inhibition, CSPG production was increased in TGFβ1-treated astrocytes as determined biochemically (Fig. 2C) and cytochemically (Supplemental Fig. S3) using an antibody recognizing 4- and 6-sulfated CS. Increased production of CSPGs in CM and cell lysates was observed after 3 days of treatment with TGFβ1. It should be noted that CS-56 positive bands were sensitive to cABC treatment and migrated faster and less diffusely on SDS-PAGE under reducing condition than non-reducing condition (Supplemental Fig. S3). However, production of laminin, a major growth permissive component of ECM, was not altered in response to TGFβ1 treatment (data not shown). More quantitatively, accumulation of CSPGs by reactive astrocytes was detected in CM using an ELISA as early as 1 day after TGFβ1 treatment (Fig. 2D). Quantitative RT-PCR revealed that transcripts of neurocan and versican were upregulated after TGFβ1 treatment (Asher et al., 2000). These data indicate that the increased production of CSPGs by reactive astrocytes is likely to be responsible for inhibition of axonal growth.
To firmly establish the involvement of CSPGs in this inhibition, we performed axonal guidance spot assays with immobilized CM derived from astrocytes (Fig. 3). Axons favored growth on PLL compared to the spot where concentrated TGFβ1-treated CM was immobilized, and this preference was abolished by cABC treatment (Fig. 3A and B), demonstrating that it is the CS GAG chains in the CM that impart neuronal guidance cues. Next, we examined the effect of GAG synthesis inhibitors on axonal growth. Astrocytes were pretreated with TGFβ1 together with xyloside or sodium chlorate, and neurons were cultured on the monolayers (Fig. 3C). Reduction of axonal growth by TGFβ1 treatment was prevented when the covalent attachment of GAG chains to the core protein was competitively inhibited by treatment of astrocytes with xylosides, or when sulfation was blocked by sodium chlorate. Together, these data provide substantial evidence that CS GAG chains produced by reactive astrocytes mediate axonal growth inhibition.
We next determined whether TGFβ1 treatment regulates the sulfation of CS GAG chains. Immunoblot analyses of CM with monoclonal antibodies 2B6 and 3B3 (specific for 4-sulfated and 6-sulfated CS GAG chains, respectively) showed substantial increases in 4-sulfation and a slight increase in 6-sulfation 3 days after TGFβ1 addition (Fig. 4A). This was confirmed quantitatively by an ELISA with another set of sulfation-specific monoclonal antibodies (MAB2030 and 2035, Fig. 4B). It is noteworthy that only 4-sulfated CS was acutely induced within 24 hours of TGFβ1 exposure, and that accumulation rates of 4-sulfated and 6-sulfated CS thereafter were similar.
The finding of this dramatic change in 4-sulfation led us to examine chondroitin sulfotransferases that are responsible for the sulfation in GalNAc. Consistent with our ELISA data, quantitative RT-PCR revealed a 3.8-fold induction in chondroitin 4-sulfotransferase 1 (C4ST1) transcript as early as 8 hours after TGFβ1 treatment that endured for 48 hours (Fig. 4C). In contrast, levels of chondroitin 6-sulfotransferase 1 (C6ST1) transcript remained unchanged (Fig. 4D). Other chondroitin sulfotransferases (chondroitin 4-sulfotransferase 2, chondroitin 6-sulfotransferase 2, and chondroitin 4,6-sulfotransferase) were not altered upon TGFβ1 treatment (data not shown). Upregulation of C4ST1 protein in reactive astrocytes was also confirmed using an anti-C4ST1 peptide antibody (Fig. 4E). The fact that the increase in C4ST1 transcript upon TGFβ1 treatment was not observed in Smad3-null astrocytes (Supplemental Fig. S3) demonstrates that the rapid change in CS sulfation is mediated by TGFβ signaling through the Smad pathway.
To investigate whether 4-sulfation of CS GAG chains is crucial for the regulation of axonal growth, loss- and gain-of-function experiments were performed. Introduction of siRNA against C4ST1 into astrocytes decreased levels of C4ST1 protein in whole cell lysates, and correspondingly reduced the accumulation of 4-sulfated CS in CM (Fig. 5A–C). Importantly, the TGFβ1-mediated increase in 4-sulfated CS and C4ST1 was blocked by C4ST1 siRNA. We then performed axonal guidance spot assays with CM from astrocytes treated with combinations of TGFβ1 and C4ST1 siRNA. CM from astrocytes treated with the combination of C4ST1 siRNA and TGFβ1 was significantly less potent than CM from astrocytes treated with TGFβ1 alone (Fig. 5D). Transfection of C4ST1 siRNA did not affect the induction of neurocan transcript by TGFβ1 (data not shown).
Because 6-sulfated GAG chains have also been suggested to be involved in the brain injury response (Properzi et al., 2005), we similarly examined the effects of alteration of C6ST1 and 6-sulfated GAG chains. Astrocytes treated with siRNA directed against C6ST1 showed a reduction of both transcript level and the production of 6-sulfated CS (Fig. 6B and D). In contrast, treatment with C6ST1 siRNA did not alter the levels of C4ST1 transcript nor 4-sulfated CS. Furthermore, TGFβ1 treatment still elicited an increase in C4ST1 mRNA and 4-sulfated CS (Fig. 6A and C). More importantly, depletion of C6ST1 did not alter the inhibitory properties of TGFβ1-treated CM in the axonal guidance assays (Fig. 6E). Moreover, C4ST1 siRNA treatment did not alter the level of the transcripts for C4ST2, C6ST2, and C46ST (data not shown). These results demonstrate the essential roles of C4ST1 and 4-sulfated CS GAG chains in the induction of repellent activity of astrocytes by TGFβ1 treatment.
Conversely, we performed gain-of-function experiments (Fig. 7). Astrocytes were transfected with either an empty vector, wild-type C4ST1 or a mutated form of C4ST1 that fails to bind 3′-phosphoadenosine 5′-phosphosulfate, and CGNs were plated on the monolayers of these astrocytes. While similar levels of exogenous proteins were expressed in astrocytes (Fig. 7A), cells expressing wild-type C4ST1 produced more 4-sulfated CSPG in the CM as compared to vector-transfected astrocytes (Fig. 7B), while astrocytes expressing the mutated form showed lower levels of 4-sulfated GAG chains. Neurons growing on astrocytes expressing C4ST1 had shorter axons than those growing on either vector-transfected astrocytes or expressing the mutant C4ST1 (Fig. 7D). HPLC analysis of CM confirmed that the production of 4-sulfated GAG was highly correlated with perturbations in C4ST1 expression (Fig. 5C and and7C).7C). We were unable to demonstrate an effect of overexperssion of C6ST1 because when exogenous C6ST1 was expressed in astrocytes, the cells looked unhealthy and survival was compromised (data not shown). Taken together, the loss- and gain-of function experiments establish a pivotal role of 4-sulfated GAG in axonal growth regulation by reactive astrocytes.
In vivo experiments confirmed that 4-sulfated CS GAG chains may be a critical determinant of CNS regenerative failure (Fig. 8). A dorsal overhemisection of the spinal cord was made in mice and we examined expressions levels of 4-sulfated and 6-sulfated GAG chains, as well as glial fibrillary acidic protein (GFAP), a well established marker of reactive astrocytes (Lemons et al., 1999; Pekny and Pekna, 2004) with specific antibodies. While we observed very low levels of immunoreactivity for 6-sulfated CS GAG chains, we found substantial staining for 4-sulfated CS GAG chains proximal to the lesion as early as 1-day post injury (Fig. 8A and D). A similar increase in GFAP immunoreactivity was observed with similar proximity to and specificity for the lesion site. Colocalization of 4-sulfated GAG and GFAP was also apparent microscopically (Fig. 8B and C). Upregulation of 4-sulfated GAG chains upon spinal cord injury was also apparent when CS disaccharides were extracted from uninjured/injured tissues and analyzed by HPLC (Fig. 8E). Thus, these data confirm a specific upregulation and deposition of 4-sulfated GAG by reactive astrocytes after CNS injury in an animal model.
CSPGs are ECM molecules that have a critical role in modulating axonal growth and guidance during development and also after nervous system injury. While much evidence has accumulated suggesting that it is the GAG chain moieties of CSPGs that are recognized by neurons, the particular features of GAG chains that signal to growing axons are still a matter of contention. In this manuscript, we present compelling evidence that this signaling is mediated through specific sulfation, specifically 4-sulfation, of the CS GAG chains. First, CS-A, but not CS-C, exhibits negative guidance cues to axons in a 4-sulfation dependent manner, with comparable efficacy to native CSPGs. Second, reactive astrocytes in culture produce more 4-sulfated CS GAG chains and knockdown of C4ST1 reduces the level of 4-sulfation in CS GAG chains, resulting in a less inhibitory ECM. Third, overexpression of C4ST1 in cultured astrocytes increases 4-sulfation and reduces their ability to support neuronal growth. Finally, 4-sulfated CS GAG chains are acutely upregulated and deposited by reactive astrocytes in an animal model of spinal cord injury. This combination of biochemical and physiological approaches synergistically demonstrate the major role of 4-sulfated GAG chains in astrocyte/neuron interactions.
The fact that CS-A, but not CS-C, repels axons highlights the exquisite structural specificity for signaling by the sulfated disaccharides that comprise CS chains. Both CSA and CS-C carry a similar charge distribution, demonstrating that these effects are not simply mediated by negative charge carried by the sulfate groups. While 6-sulfation of CS GAG chains has been reported to correlate with axonal inhibition (Properzi et al., 2005), we did not find any inhibitory action of CS-C in our axonal guidance assays, and siRNA-based depletion of C6ST1 in reactive astrocytes showed no effect on axonal guidance
Only a small change in 4-sulfation significantly alters the potency of CS-A to impart neuronal guidance, suggesting that subsets of sulfation are critical determinants of function. This notion is supported by the finding that the biological activity of CS-A is eliminated after only a short duration of treatment with cABC that digests as little as 2% of the GAG. Conversely, only a small percentage of 4-sulfation was reported to increase in vivo following injury, even though 4-sulfated disaccharides are the predominant species in the normal brain (Gris et al., 2007; Mitsunaga et al., 2006; Properzi et al., 2005). These data suggest that it is not the level of 4-sulfation per se that contributes to GAG chain signaling. It has been proposed that distinct motifs of sulfation (a "sulfation code") along the polysaccharide chain in heparan sulfate encode information required for substrate binding and growth regulation (Bülow and Hobert, 2004; Holt and Dickson, 2005). Although heparan sulfate and chondroitin sulfate are structurally different, our findings may suggest the presence of a "sulfation code" in CS GAG chains that exhibits negative guidance cues to axons and inhibit axonal growth.
The direction and rate of axonal extension can be independently modulated by ECM (Powell et al., 1997). The axonal guidance spot assays used in this study focus simply upon axonal guidance: axons growing on the PLL substrate turn as they encounter CS-A, and continue to extend along the interface (data not shown). Similar behavior is observed in vivo as growing axons encounter the CSPG-rich glial scar (Davies et al., 1997). In contrast, axonal growth depends upon both cell adhesion and neurite initiation/extension, and alterations in either of these conditions will result in measurable changes. Our intriguing discovery is that 4-sulfation of CS GAG chains both alters axonal direction and limits the rate of axonal extension.
Paradoxically, tissues that express CSPGs do not always exclude the entry of axons, and in some cases CSPG staining coincides with developing and regenerating axon pathways (Bicknese et al., 1994; McAdams and McLoon, 1995). Axonal extension during development and after injury to the adult CNS is a balance of inhibitory and promotional cues in the local environment consisting of several ECM molecules, cell adhesion molecules, and growth factors (Lu et al., 2007; McKeon et al., 1995; Walsh and Doherty, 1996). In addition, changes in sulfation of CS GAG chains are likely to contribute to the determination of the success or failure of axonal regeneration. Several in vitro studies suggest that CSPGs can promote rather than inhibit neurite outgrowth (Faissner et al., 1994; Fernaud-Espinosa et al., 1994; Garwood et al., 1999). These promotional effects have been attributed to the “oversulfated” chondroitin sulfates: CS-D (disulfated at the C2 position of GlcA and C6 position of GalNAc) and CS-E (disulfated at the C4 and C6 positions of GalNAc), both of which stimulate neurite growth in culture (Deepa et al., 2002; Gama and Hsieh-Wilson, 2005; Gama et al., 2006; Nadanaka et al., 1998). Axonal growth promotion has also been observed with an artificial tetrasaccharide with 4,6-sulfation, suggesting that a short stretch of sulfated GAG chains are sufficient to promote neurite outgrowth (Gama et al., 2006). Interestingly, when we used CS-D and CS-E in our axonal guidance assays, we did not observe any positive haptotactic effects of these sugars. Because oversulfated CS chains have been shown to bind several different growth promoting factors and cytokines (Deepa et al., 2002; Shipp and Hsieh-Wilson, 2007), the growth-promotional actions of these CS sugars may be indirect.
In the developing brain, astrocytes are a preferred substrate for axonal growth and neuronal migration, while reactive astrocytes in the injured brain are detrimental to neuronal regeneration. The major difference in this functional shift is the increased production of sulfated proteoglycans by reactive astrocytes. Using a physiologically relevant system, we found that modulation of the sulfation in astrocytic CSPGs changes the interaction between astrocytes and neurons in vitro. Combined with our observation that 4-sulfated CSPGs are robustly and rapidly deposited within CNS lesions in animals, these findings suggest that modulation of sulfation in CSPGs serves as a signal to restrict axonal regrowth and may be an important new therapeutic direction for regenerative biomedicine.
Cultures of dissociated mouse CGNs were prepared from C57BL/6 mice (P5-8) as described previously (Levi et al., 1984; Romero et al., 2003). Dissociated cells were cultured in Neurobasal-A medium containing B27 supplement and 25 mM KCl. In co-culture experiments, dissociated CGNs were plated at a density of 6 × 104 cells/well onto a confluent monolayer of astrocytes in 24-well plates (see below). When neurons were cultured in CM, 2% (v/v) of B27 supplement was added. Primary cortical neuron cultures were prepared from E16-E18 mouse embryos as previously described (Dulabon et al., 2000).
Primary cultures of cerebral cortical astrocytes were prepared from newborn C57BL/6 mice and Smad3 null mice (Wang et al., 2007) (P1–2) as previously described (Petroski et al., 1991). Confluent cultures of astrocytes were treated with TGFβ1 (10 ng/ml, R&D systems) in the absence of serum for 7 days and dissociated CGNs were plated on the monolayers. Two days after plating, cells were fixed and stained with anti βIII-tubulin antibody (Sigma), followed by the incubation with FITC-anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories). When astrocytes were treated with inhibitors, monolayers of astrocytes were incubated with TGFβ1 in combination with those inhibitors for 72 hours, after which neurons were plated on top and allowed to grow for 48 hours before analysis.
Axonal guidance spot assays were performed as described previously (Meiners et al., 1999). To quantify the behavior of axons, an interface between PLL and sample was created by placing a 5 µl drop of chicken CSPG (Millipore, 12.5 ng/spot) or CS GAG chains (Seikagaku, Japan) together with Texas Red in the center of a PLL-coated glass cover slip. Texas Red was used to visualize the interface and was used alone for negative control experiments. Dissociated CGNs were seeded onto the cover slips at a density of 6 × 104 cells/well and cultured for 2 days. Cells were fixed and stained with anti βIII-tubulin antibody (Sigma), followed by the incubation with FITC-anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories). Fluorescence images were acquired on a Nikon TE2000 inverted microscope equipped with a CCD camera (Orca-ER, Hamamatsu) driven by Metamorph imaging software (Universal Imaging Corp.). Only single, non-fasciculated axons within 10 µm of the protein-PLL interface were considered for the analysis. In addition, only axons growing toward the immobilized sample were counted and no axon whose soma was sitting on the interface was scored. Each experiment was performed in triplicate. For treatment with cABC (Seikagaku), samples were digested with 10 mU of the enzyme at 37°C for 3 hours. Chondro-4-sulfatase (Seikagaku) digestion was carried out with 8 mU of the enzyme in 0.1M ammonium acetate buffer (pH 7.0) at 37°C for 4 hours, followed by the inactivation of the enzyme at 95°C for 5 minutes.
Axonal outgrowth assays were performed as described previously (Meiners et al., 1999). Axonal length was measured using the ImageJ program (available at: http://rsb.info.nih.gov/nih-image/). A sample of 100 neurons with processes equal to or greater than one cell soma was considered for each condition. The total length of each primary process was measured for each neuron. For some experiments, relative axonal length was also obtained using a stereological technique from a large sample of neurons (Rønn et al., 2000). When neurons were co-cultured with astrocytes overexpressing C4ST1, astrocytes were first transfected with appropriate DNA constructs, followed by replacement of the media one day after transfection and an additional one day culture, and neurons were then plated onto the monolayer of astrocytes. Cells were fixed, stained with anti βIII-tubulin antibody, and relative axonal length was measured as described above. Astrocyte-derived conditioned media and cell lysates were collected 2 days after transfection for immunoblot and disaccharide composition analysis.
Computed values were compared between the different conditions using either Student’s t-test or one-way ANOVA, as appropriate.
The cDNA for chondroitin 4-sulfotransferase 1 (C4ST1) was obtained by RT-PCR from mouse astrocyte RNA using the following primers; 5’- TAGAATTCAC TAGTATGAAG CCGGCGCTGC TGGAAG-3’ and 5’- ATGAATTCCA CTCGAGTCCA ACTTCAGGTA GTTTGG-3’. PCR product was digested with EcoRI, followed by subcloning into the EcoRI site of pDsRed2-N1 (BD Biosciences) and pTracer-EF/V5His (Invitrogen). An inactive form of C4ST1 was generated by the introduction of mutations (R186A, S194A) into the putative PAPS binding site with QuickChange (Stratagene, CA) using the following primers; 5’- GTTCCTGTTC GTGGCTGAGC CCTTCGAGAG G-3’ and 5’-GAGCCCTTCG AGAGACTAGT GGCTGCCTAC CGCAAC-3’. The cDNA for mouse chondroitin 6-sulfotransferase 1 (C6ST1) was obtained by RT-PCR using the following primers; 5’-ATGAATTCAC TAGTATGGAG AAAGGACTCG CTTTGC -3’ and 5’-AAAAGCTTCT ACGTGACCCA GAAGGTGC -3’. PCR product was digested with EcoRI/HindIII, followed by subcloning into the EcoRI/HindIII sites of pDRed2-N1 (BD Biosciences).
Transient transfection was performed using the Nucleofector (Amaxa GmbH, Cologna, Germany) with a protocol specifically designed for mouse astrocytes. After transfection, astrocytes were plated on a 35 mm dish and allowed to become confluent.
CSPGs in conditioned media (CM) and cell lysates derived from astrocyte cultures were separated on SDS-PAGE under reducing conditions and examined with immunoblot analyses as described previously (Katagiri et al., 2000). When CM was concentrated with a Centricon 100 (Millipore), a protease inhibitor cocktail (Calbiochem) was added to prevent protein degradation. Samples to be incubated with the 2B6, 3B3 (Seikagaku), MAB2030, or MAB2035 (Millipore) antibodies required prior digestion with cABC (10 mU/ml, 37°C for 3 h) to expose the antigen. For immunoblotting of C4ST1, we generated a custom chicken anti-C4ST1 peptide antibody (Gallus Immunotech Inc.) against the peptide sequence RRQRKNATQEALRKGDDVKC. HeLa cell lysates expressing recombinant C4ST1 with a V5 epitope tag (pTracer-C4ST1) were used as positive controls for immunoblot. Chicken CSPGs (Millipore) used in this study contained neurocan and phosphocan, confirmed by cABC digestion and tryptic digestion, followed by mass spectrometry, but we did not exclude the presence of other core proteins (data not shown).
TGFβ1-treated astrocytes cultured for 7 days on glass cover slips were rinsed with DMEM and incubated with CS-56 (Sigma) for 30 minutes at 4°C, followed by incubation with FITC-anti-mouse IgM antibody (Jackson ImmunoResearch Laboratories). Cells were then fixed and incubated with rabbit anti-GFAP antibody followed by Rhodamine-anti-rabbit IgG antibody.
Chemically synthesized siRNA targeting C4ST1, C6ST1, and scramble siRNA (as a negative control) were obtained from Dharmacon (siGENOME™ SMARTpool®). Transient transfection into primary cultured astrocytes with 3 mg siRNA was carried out using the mouse astrocyte Nucleofector kit (Amaxa). Transfection efficiency was more than 80% based the simultaneous transfection of pmaxGFP™. Medium was replaced with DMEM one day after transfection and the cells were cultured for two more days. The cells were then treated with TGFβ1 for 24 hours and CM was collected for axonal guidance spot assays, immunoblot, and ELISA.
Total RNA was isolated from cultured astrocytes with the Absolutely RNA purification kit (Stratagene). Genomic DNA was removed by DNaseI treatment following the manufacturer’s protocol. RNA was reverse transcribed using SuperScript III (Invitrogen) and real-time PCR was performed on a Chromo4 (MJ Research) with DyNAmoT™ HS SYBR Green qPCR kit (MJ Research). PCR conditions consisted of a 15 minutes hot start at 95°C, followed by 45 cycles of 15 s at 94°C, 15 s at 57°C, and 25 s at 72°C. All samples were run in triplicate and results were normalized to the level of GAPDH. The primer sequences are as follows: C4ST1; 5’- GAAGAGGCTC ATGATGGTCC -3’ and 5’- GAGAGAGTAG ACCGTCTG CC -3’, C6ST1; 5’- GGATTCCACC TTTTCCCATCTG -3’ and 5’- TGCCCTGCTG GTTGAAGAAC -3’, and GAPDH; 5’- AAGGTGGTGA AGCAGGCATC TG -3’ and 5’- TGGGTGGTCC AGGGTTTCTT AC -3’. cDNAs for C4ST1, C6ST1, and GAPDH were used as a template for PCR to obtain standard curves.
Relative CSPG amounts were measured by ELISA. Briefly, 96 well microtiter plates (Immulon 4; Dynex Technologies) pretreated with poly-L-Lysine were coated with CM of astrocytes treated with or without TGFβ1. After blocking, appropriate antibodies were incubated, followed by incubation with anti-mouse antibody F(ab’)2 fragment conjugated with HRP (Abcam). Binding was measured with a microplate reader (Labsystems Multiskan, MCC/340) using SureBlue TMB Microwell Peroxidase Substrate (KPL) as a substrate.
All experiments strictly adhered to the NIH guidelines on the care and use of animals in research. Adult mice (8–12 weeks old) were deeply anesthetized with ketamine-xylazine (100 and 14 mg/kg, respectively). A laminectomy was performed at the level of T12-L1 and the spinal cord was exposed. A dorsal overhemisection was made at T12. After the injury, the subcutaneous tissue and skin were sutured in layers. One day after surgery, animals were anesthetized then perfused intracardially with PBS, followed by 4% paraformaldehyde. Spinal cords were removed and frozen. Sagittal serial sections were cut on a cryostat (15 µm) and processed for histological analyses. Monoclonal antibodies, LY111, MC21C (both Seikagaku), were used to detect changes in 4-sulfation and 6-sulfation in intact CS GAG chains, respectively, and polyclonal anti-GFAP antibody (Dako) was to visualize reactive astrocytes, followed by the incubation with FITC-conjugated anti-mouse µ chain antibody (Abcam) and Alexafluor 633-conjugated anti-rabbit Ig antibody (Molecular Probes). Fluorescent images were acquired with a confocal laser scanning microscope (Leica SP1, Leica, Germany).
Disaccharide composition analysis was performed essentially as described previously (Kinoshita and Sugahara, 1999) with minor modification. Briefly, CS oligosaccharides in 0.1M ammonium acetate buffer (pH 7.0) were treated with cABC as described above and lyophilized. Derivatization of the oligosaccharides with 2-aminobenzamide (2AB, Sigma) was carried out with 5 µl of 0.35 M 2AB/1.0M NaCNBH4 /30 % acetic acid in DMSO at 65°C for 2 hours. Fluorescently tagged oligosaccharides were separated by HPLC on an amine-bound silica PA03 column (Waters). The HPLC system was equilibrated with solvent A (15 mM ammonium phosphate containing 5 % methanol) and solvent B (1.5 M ammonium phosphate containing 5 % methanol). At a uniform flow rate of 0.75 ml/min, a gradient was developed by holding solvent B at 0 % for 5 minutes, then increasing from 0 to 18 % over 14 minutes and changing from 18 to 50 % over 11 minutes. Separation was monitored using a L-7485 fluorescence detector (Hitachi Ltd, Japan) with excitation and emission wavelengths of 330 and 420 nm, respectively. When CM was used as a source for CSPGs, concentrated samples were digested with proteinase K extensively, followed by cABC treatment. 3′-Sialyl-N-acetyllactosamine (Dextra Laboratories, UK) was added to GAG-containing fraction as an internal control. Disaccharide composition analysis was performed as described above.
Disaccharide composition of CS chains in the spinal cord sections on the glass slides were determined as described previously (Mitsunaga et al., 2006). Briefly, coronal cryosections of spinal cords were treated with cABC (25 mU/ml) for 16 hours at 37°C together with 3′-Sialyl-N-acetyllactosamine on the glass slides. The solution was recovered and disaccharide fractions were enriched by size exclusion chromatography (Superdex Peptide10/300 GL, GE Healthcare) in 150 mM ammonium bicarbonate at a flow rate of 0.3 ml/min. Separation was monitored using a L-7400 UV detector (Hitachi Ltd, Japan) with absorbance of 232 nm. Purified disaccharides were derivatized with 2-AB.
Partial digestion of CS-A was performed with cABC (10 mU/ml) at room temperature and the digestion was monitored by the absorbance at 232 nm. The digestion degree was calculated based on the comparison of the absorbance measured at 232 nm at the time of aliquot removal and the one at the time of reaction completion. After heat inactivation of the enzyme, chondroitinase-treated CS-A was precipitated with ethanol and subjected to axonal guidance spot assays.
Video 1 shows that the growing axons from mouse DRG turn at the interface between PLL and CSPG and continue to extend along the interface.
Fig. S1 shows disaccharide composition analysis of CS GAG chains with HPLC.
Fig. S2 shows that Cerebral cortical neurons were repelled by CS-A as well as CSPG.
Fig. S3 shows that increased production of CSPGs by reactive astrocytes is responsible for reduced neuronal growth.
We thank Drs. M.V. Sofroniew for spinal cord tissues, and R. Adelstein, J. Sellers, N. Epstein, and Z.H. Sheng for critical comments; Dr. S. Wen, Dr. T. Laabs, K. Vartanian, and Y. Tailor for technical support. We are grateful to Dr. R. F. Shen and Dr. C.A. Combs for help with data collection at Proteomics Core Facility and Light Microscopy Core Facility in National Heart, Lung, and Blood Institute, NIH.