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.