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Sulfated glycans in network rewiring and plasticity after neuronal injuries
Neuroscience Research 78 (2014) 50–54
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Review article
Sulfated glycans in network rewiring and plasticity after neuronal injuries Kenji Kadomatsu ∗ , Kazuma Sakamoto Department of Biochemistry, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan
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Article history: Received 28 March 2013 Received in revised form 24 September 2013 Accepted 27 September 2013 Available online 21 October 2013 Keywords: Neuronal injury Keratan sulfate Condroitin sulfate
a b s t r a c t Biopolymers in the human body belong to three major classes: polynucleotides (DNA, RNA), polypeptides (proteins) and polysaccharides (glycans). Although striking progress in our understanding of neurobiology has been achieved through a focus on polypeptides as the main players, important biological functions are also expected to be attributable to glycans. Nonetheless, the significance of glycans remains largely unexplored. In this review, we focus on the roles of sulfated glycans. Axonal regeneration/sprouting after injuries does not easily occur in the adult mammalian central nervous system. This is due to the low intrinsic potential of regeneration and the emerging inhibitory molecules. The latter include the sulfated long glycans chondroitin sulfate (CS) and keratan sulfate (KS). Enzymatic ablation of CS or KS, and genetic ablation of KS promote functional recovery after spinal cord injury. Interestingly, the combination of CS and KS ablations exhibits neither additive nor synergistic effects. Thus, KS and CS work in the same pathway in inhibition of axonal regeneration/sprouting. Furthermore, CS has been implicated in neural plasticity as a functional component of the perineuronal nets surrounding inhibitory interneurons. Elucidation of the mechanisms of action for KS and CS will pave the way to treatments to promote network rewiring and plasticity after neuronal injuries. © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
Contents 1. 2. 3. 4. 5. 6.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CSPGs as a major inhibitor of axonal regeneration/sprouting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CSPGs limit experience-dependent neural plasticity and synaptic plasticity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Receptors for CSPGs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . KSPGs as a major inhibitor of axonal regeneration/sprouting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . KS and CS work in the same pathway in the inhibition of axonal regeneration/sprouting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction The functional neuronal circuits are formed during development as well as after injuries, depending not only on genetic information but also on plastic remodeling in response to environmental information. Gradients of extracellular cues induce second messengers, such as calcium and cyclic nucleotides, and transduce attractive and repulsive signals by steering growth cones through the creation of localized imbalances between exocytosis and endocytosis (Kolodkin and Tessier-Lavigne, 2011; Tojima et al., 2011).
∗ Corresponding author. Tel.: +81 52 744 2059. E-mail address: [email protected] (K. Kadomatsu).
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Since axons in the adult mammalian central nervous system (CNS) lack regenerative capacity, it is an important question whether new growth cones generated after injury use the same strategy for regeneration as embryonic neurons employ, and how the injured axons compromise the low intrinsic activity of regeneration (Bradke et al., 2012; Harel and Strittmatter, 2006). Furthermore, the emerging inhibitors after injuries are strong barriers for regeneration. The inhibitors can be categorized into three classes: canonical axon guidance molecules, myelin-derived inhibitors, and chondroitin sulfate proteoglycans (CSPGs) (Giger et al., 2010; Harel and Strittmatter, 2006; Silver and Miller, 2004). Axon guidance molecules include semaphorins, ephrins, netrins and slit. These molecules are expressed upon injury and induce growth cone
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Fig. 2. Schematic presentation of CS and KS induction and the effects of their ablation. CS and KS are expressed at or around the site of injury. Axonal regeneration/sprouting hardly occur after neuronal injuries. However, if CS or KS are digested, axonal regeneration/sprouting is enhanced and functional recovery is promoted. Fig. 1. Structure of proteoglycan. (A) Structure of proteoglycan. Proteoglycan consists of core protein and glycosaminoglycans (GAGs). GAG is a long sugar chain composed of repeating disaccharide units. (B) Biosynthesis of KS. KS synthesis is accomplished by a cycle of GlcNAc transfer, GlcNAc sulfation and Gal transfer that are mediated by the enzymes 3GnT7, GlcNAc6ST and 4GalT4, respectively. After elongation of the KS chain to some extents, Gal is sulfated by the enzyme KSGal6ST. This oversulfated form of KS is recognized by the antibody 5D4. GlcNAc6ST-1 is responsible for GlcNAc sulfation for the production of 5D4-resposive KS in the brain. Therefore, the deficiency of GlcNAc6ST-1 results in loss of 5D4-resposive KS in the brain.
collapse. Blocking agents against these inhibitors could be beneficial for the functional recovery after injuries, such as spinal cord injury (SCI) (Hata et al., 2006; Kaneko et al., 2006). The myelinderived inhibitors consist of Nogo, myelin-associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein (OMgp). These glycoproteins strongly inhibit neurite outgrowth in vitro. However, the significance of their in vivo functions is still in debate. Thus, triple knockout mice of these three genes have been reported not to exhibit enhanced regeneration of axonal tracts after SCI, while another report showed that the triple knockout mice exhibited greater axonal growth and improved functional recovery (Cafferty et al., 2010; Lee et al., 2010). Silver and colleagues reported in 1990 that axons from the E9 chick dorsal ganglia will not cross a strip of proteoglycans coated on nitrocellulose (Snow et al., 1990a). However, digestion of chondroitin sulfate (CS) abolishes the inhibitory activity of proteoglycans. These findings triggered studies on chondroitin sulfate proteoglycans (CSPGs) as inhibitors of axonal regeneration/sprouting. Glycans consisting of repeating disaccharide units are named glycosaminoglycans (GAGs). CS is a sulfated GAG, the disaccharide units of which are composed of GlcA and GalNAc. CS chains are covalently attached to a core protein, and this whole molecule is generally called CSPG (Fig. 1). It is now accepted that CSPGs act not only as inhibitors of axonal regeneration/sprouting but also as regulators restricting synaptic plasticity and experiencedependent neural plasticity (Bradbury et al., 2002; Frischknecht et al., 2009; Gogolla et al., 2009; Moon et al., 2001; Pizzorusso et al., 2002). We have recently found that the functions of keratan sulfate (KS) and CS merge in the same pathway in inhibition of axonal regeneration/sprouting, and that both sugar chains are essential for the inhibition (Imagama et al., 2011; Ito et al., 2010; Tauchi et al., 2012; Zhang et al., 2006). Here, we overview the currently known biological activities of CSPGs in network rewiring and plasticity after neuronal injuries. Then, we show the functions of KSPGs and discuss the relationship between KS and CS.
2. CSPGs as a major inhibitor of axonal regeneration/sprouting Trauma to the central nervous system (CNS) leads to rupture of the blood–brain barrier, which in turn allows infiltrations of macrophage from the blood and fibroblasts from the meninges, and consequently induces glial scar formation (Silver and Miller, 2004). The glial scar consists of reactive astrocytes and other cellular components as well as extracellular molecules including CSPGs. Round, swollen axonal ends with multiple vacuoles are often found in the glial scar, and are called dystrophic endballs. Dystrophic endballs end in the CSPG deposition in the glial scar, and persist there for at least 9.5–13 weeks after injury (Davies et al., 1999; Li and Raisman, 1995). The formation of dystrophic endballs can be recapitulated in vitro if axons of adult dorsal root ganglion cells are forced to grow against a gradient of CSPGs (Tom et al., 2004). Under this condition, axons cannot extend well. Neurite outgrowth on the substratum evenly coated with CSPGs is also inhibited, and this inhibition is released by the treatment with the CS-degrading enzyme chondroitinase ABC (C-ABC). Consistent with this, C-ABC promotes axonal regeneration after nigrostriatal tract transaction, sprouting of spared fibers in the cuneate nucleus after cervical spinal cord injury (SCI), and functional recovery after SCI (Bradbury et al., 2002; Massey et al., 2006; Moon et al., 2001) (Fig. 2). Therefore, the CS chains of the CSPG moiety seem to be principal actor for the CSPG-mediated inhibition of axonal regeneration/sprouting. 3. CSPGs limit experience-dependent neural plasticity and synaptic plasticity Special extracellular matrix structures, the so-called perineuronal nets (PNNs), are found around a subset of parvalbuminpositive interneurons. PNNs consist of hyaluronan, CSPGs (aggrecan, versican, brevican, neurocan), and tenacin R. Although the precise underlying mechanisms remain elusive, PNNs have been implicated in neural plasticity. For example, ocular dominance plasticity occurs during a postnatal critical period (19–32 postnatal days in mice; 12–36 months in humans), but not in adults. CS digestion with C-ABC restores the ocular dominance plasticity in adult animals (Pizzorusso et al., 2002). Surprisingly, transgenic mice that show an infant-type sulfation pattern of CS also manifest ocular dominance plasticity in adults, suggesting the importance of sulfation modes of CS in PNNs in this plasticity (Miyata et al., 2012). These findings are of clinical importance, since ocular dominance
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plasticity is related to monocular amblyopia. PNNs are also essential for the protection of fear memory from erasure in adult animals. Thus, if the CS in PNNs in the amygdala is digested by C-ABC, fear memory becomes susceptible to erasure. Traumatic memories can be erased by extinction during early development, but PNNs may close this critical period and form erasure-resistant fear memories in adults (Gogolla et al., 2009). Interestingly, other molecules involved in the inhibition of axonal regeneration/sprouting, such as Nogo receptors (NogoRs) and PirB (both are known as receptors for myelin-derived inhibitors), also limit ocular dominance plasticity (McGee et al., 2005; Syken et al., 2006). Furthermore, Nogo receptors restrict spine formation and dendrite branching (Wills et al., 2012). In addition to experience-dependent plasticity, e.g., ocular dominance plasticity and fear memory erasure, CS and hyaluronan have been implicated in synaptic plasticity. Lateral diffusion of glutamate receptors is a critical step for the accumulation of these receptors in the post-synapses, and thus contributes to the synaptic plasticity of excitatory neurons. Digestion of CS or hyaluronan promotes lateral diffusion of glutamate receptors and long term potentiation (Frischknecht et al., 2009). Furthermore, CS is also involved in dendritic spine dynamics. Thus, C-ABC enhances the motility of dendritic spines and induces the appearance of spine head protrusions in a glutamate receptor-independent manner. CSPGs located around dendritic spines are sufficient to restrict spine dynamics independently of PNNs, since microinjection of C-ABC close to dendritic segments can induce spine remodeling (Orlando et al., 2012).
4. Receptors for CSPGs Several receptors for CS have been postulated. These include PTP, LAR, NogoR1, NogoR3, and contactin-1 (Coles et al., 2011; Dickendesher et al., 2012; Fisher et al., 2011; Mikami et al., 2009; Shen et al., 2009). Blocking or ablation of PTP, LAR, NogoR1 and NogoR3 restores the neurite outgrowth inhibited by CSPGs and promotes axonal regeneration/sprouting in vivo. Contactin-1 binds to CS-E (E indicates a type of sulfation), and is involved in CS-Emediated neurite outgrowth under some conditions (Mikami et al., 2009).
Regarding the mechanisms of CS action, it is worthy of note that CSPGs and heparan sulfate proteoglycans (HSPGs) show distinct localization and functions. HSPGs are located on the surface of axons, while CSPGs are rich in extracellular spaces. Structural analysis of PTP has revealed that HSPGs bind to PTP and promote oligomerization of PTP on axons (Coles et al., 2011). Once the production of CSPGs in the extracellular space is enhanced after injuries, CSPGs bind to PTP. As a consequence, the PTP oligomers may be disrupted and PTP may be turned on. In this way, HSPGs and CSPGs work as a switch in response to injury. The transmembrane protein semaphorin 5A (Sema5A) is also present at the intersection between HSPGs and CSPGs. The extension of axons of the fasciculus retroflexus is promoted by Sema5A through binding of HSPGs on the axons, while CSPGs precisely localized in the extracellular space convert Sema5A from an attractive to an inhibitory guidance cue (Kantor et al., 2004).
5. KSPGs as a major inhibitor of axonal regeneration/sprouting KS is expressed in the glial roof plate in the spinal cord during E13.5-15.5 in a temporal- and spatial-specific manner (Snow et al., 1990b). The commissural and dorsal column axons never cross the roof plate, suggesting that KS functions as a guidance cue for these axons. As described above, axons from the dorsal root ganglia will not cross a strip of proteoglycans coated on nitrocellulose. This inhibition is abolished by digestion of not only CS but also KS (Snow et al., 1990a). Furthermore, mossy fibers originate from granule cells of the dentate gyrus and synapse on the proximal apical dendrites of CA pyramidal neurons within a narrow band called the stratum lucidum. Treatment with keratanase, but not C-ABC, leads to the misrouting of mossy fibers in rat hippocampal slice cultures (Butler et al., 2004). Interestingly, mice deficient in PTP, a receptor for CS, show a similar phenotype, o.e., increased collateral branching of mossy fiber, suggesting that PTP might work as a KS receptor (Horn et al., 2012). These findings suggest that KS may also be vital for axonal guidance and/or axonal regeneration/sprouting. In line with this, KS expression is upregulated at SCI lesions (Imagama et al., 2011; Ito et al., 2010; Jones and Tuszynski, 2002) (Fig. 2).
Fig. 3. KS expression and in vivo neurite growth after a cortical stab wound. CS expression is induced at the site of a stab wound in both wild-type and GlcNAc6ST-1 KO mice. In contrast, KS expression is only induced in wild-type mice, but not in GlcNAc6ST-1 KO mice. As a result, neurite outgrowth in vivo that is represented by phosphoneurofilament staining is enhanced in GlcNAc6ST-1 KO mice. These data suggest that KS is an essential inhibitor of axonal regeneration/sprouting after neuronal injuries. Modified from Zhang et al. (2006).
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Fig. 4. Effects of KS and CS ablation on neurite growth in vitro and on functional recovery after spinal cord injury. In vitro neurite outgrowth assay indicates that proteoglycan (PG) inhibits neurite outgrowth while CS-digestion (C-ABC) or KS-digestion (K-II) reverses this inhibition. Interestingly, the combination of CS- and KS-digestion shows neither additive nor synergistic effect. Consistent with these data, CS- or KS-digestion promotes motor function recovery after SCI. But, the combination does not show additive or synergistic effect. Modified from Imagama et al. (2011).
KS is made up of repeating disaccharide units consisting of Gal and GlcNAc. For the generation of a KS chain, GlcNAc is sulfated at the C6 position, Gal is transferred and GlcNAc is further transferred to the chains. Therefore, GlcNAc sulfation is essential for the KS chain elongation (Fig. 1). This sulfation is mediated by GlcNAc6ST (GlcNAc 6-O-sulfotransferase). Four and five GlcNAc6STs have been identified in mice and humans, respectively, and are implicated in various biological activities (Uchimura and Rosen, 2006). Among them, we cloned mouse GlcNAc6ST-1 and generated its deficient mice (Uchimura et al., 1998, 2004). A developmentally regulated expression of KS can be determined by the oversulfated KS-specific antibody 5D4, but this expression is completely abolished in GlcNAc6ST-1 KO mice (Zhang et al., 2006). While wild-type mice show induction of KS and CS expression at stab wound sites in the cerebral cortex, GlcNAc6ST-1 KO mice exhibit CS induction, but not KS, in response to this trauma. To our surprise, GlcNAc6ST-1 KO mice show the enhanced neurite outgrowth in vivo at the stab wound site even if CS expression is induced there. These data suggest that KS inhibits neurite growth independently of CS (Zhang et al., 2006) (Fig. 3). GlcNAc6ST-1 KO mice also show significantly better functional recovery as well as axonal regeneration/sprouting after SCI (Ito et al., 2010). Consistent with this, chick brain-derived proteoglycans that are commonly used as representative proteoglycans to inhibit neurite growth in vitro contain not only CSPGs but also KSPGs (Imagama et al., 2011). KS digestion of chick brain-derived proteoglycans reverses the inhibition of neurite growth in vitro. Local administration of keratanase II, an enzyme that specifically digests KS, to the injured site promotes the recovery of motor and sensory functions after SCI (Imagama et al., 2011). 6. KS and CS work in the same pathway in the inhibition of axonal regeneration/sprouting KS digestion and CS digestion show comparable effects on functional recovery in vivo and neurite growth in vitro, but the combination of these digestions does not exert additive or synergistic effects (Fig. 4). These findings provide new insights into the mechanisms of proteoglycan-mediated inhibition of axonal regeneration/sprouting. Both KS and CS are essential for this inhibition. We speculate that KS and CS might share a complex of receptors, each of which individually binds CS or KS. Alternatively, the receptors might exchange cross-talk to mediate the same common intracellular signaling pathway. Considering that CSPGs are also
involved in neural plasticity, the elucidation of KS and CS signaling would have a major impact on our understanding of neuronal network rewiring and plasticity after injury and in treating disorders such as SCI, stroke and neurodegenerative diseases. However, in order to uncover the biological significance of KS and CS, an integrative and comprehensive understanding of the mechanisms underlying network rewiring and plasticity after neuronal injuries may be necessary. For example, unlike adult CNS neurons, neural stem cell-derived neurons extend axons across the lesion and over long distances. These neurons form electrophysiological relays across the lesion, which leads to functional recovery. Human neural stem cell lines show similar activities (Lu et al., 2012). Another report had demonstrated that the combination of a peripheral nerve autograft and C-ABC promotes the plasticity of spared tracts and long-distance regeneration of essential pathways, leading to a remarkable recovery of diaphragmatic function (Alilain et al., 2011). Furthermore, C-ABC in combination with rehabilitation has been shown to promote functional recovery in acute SCI as well as chronic SCI (Garcia-Alias et al., 2009; Wang et al., 2011). It has also been reported that cells expressing the CSPG NG2 stabilize dystrophic endballs rather than inhibiting regenerating sensory axons in the inhibitory environment of the glial scar (Busch et al., 2010). The continued clarification of these issues, i.e., the distinct intrinsic regeneration potentials of neurons, the cross-talk between proteoglycans and rehabilitation, and the formation of dystrophic endballs, should help to promote studies on KS and CS. Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific Research on Innovative Areas (No. 23110002 to K.K.) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan, Grant-in-Aid for Research Activity Start-up (No. 24890084 to K.S.), and by funds from the Global COE program, MEXT, to Nagoya University. References Alilain, W.J., Horn, K.P., Hu, H., Dick, T.E., Silver, J., 2011. Functional regeneration of respiratory pathways after spinal cord injury. Nature 475, 196–200. Bradbury, E.J., Moon, L.D., Popat, R.J., King, V.R., Bennett, G.S., Patel, P.N., Fawcett, J.W., McMahon, S.B., 2002. Chondroitinase ABC promotes functional recovery after spinal cord injury. Nature 416, 636–640. Bradke, F., Fawcett, J.W., Spira, M.E., 2012. Assembly of a new growth cone after axotomy: the precursor to axon regeneration. Nature Reviews. Neuroscience 13, 183–193.
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Neuroscience Research journal homepage: www.elsevier.com/locate/neures
Review article
Sulfated glycans in network rewiring and plasticity after neuronal injuries Kenji Kadomatsu ∗ , Kazuma Sakamoto Department of Biochemistry, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan
a r t i c l e
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Article history: Received 28 March 2013 Received in revised form 24 September 2013 Accepted 27 September 2013 Available online 21 October 2013 Keywords: Neuronal injury Keratan sulfate Condroitin sulfate
a b s t r a c t Biopolymers in the human body belong to three major classes: polynucleotides (DNA, RNA), polypeptides (proteins) and polysaccharides (glycans). Although striking progress in our understanding of neurobiology has been achieved through a focus on polypeptides as the main players, important biological functions are also expected to be attributable to glycans. Nonetheless, the significance of glycans remains largely unexplored. In this review, we focus on the roles of sulfated glycans. Axonal regeneration/sprouting after injuries does not easily occur in the adult mammalian central nervous system. This is due to the low intrinsic potential of regeneration and the emerging inhibitory molecules. The latter include the sulfated long glycans chondroitin sulfate (CS) and keratan sulfate (KS). Enzymatic ablation of CS or KS, and genetic ablation of KS promote functional recovery after spinal cord injury. Interestingly, the combination of CS and KS ablations exhibits neither additive nor synergistic effects. Thus, KS and CS work in the same pathway in inhibition of axonal regeneration/sprouting. Furthermore, CS has been implicated in neural plasticity as a functional component of the perineuronal nets surrounding inhibitory interneurons. Elucidation of the mechanisms of action for KS and CS will pave the way to treatments to promote network rewiring and plasticity after neuronal injuries. © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
Contents 1. 2. 3. 4. 5. 6.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CSPGs as a major inhibitor of axonal regeneration/sprouting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CSPGs limit experience-dependent neural plasticity and synaptic plasticity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Receptors for CSPGs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . KSPGs as a major inhibitor of axonal regeneration/sprouting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . KS and CS work in the same pathway in the inhibition of axonal regeneration/sprouting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction The functional neuronal circuits are formed during development as well as after injuries, depending not only on genetic information but also on plastic remodeling in response to environmental information. Gradients of extracellular cues induce second messengers, such as calcium and cyclic nucleotides, and transduce attractive and repulsive signals by steering growth cones through the creation of localized imbalances between exocytosis and endocytosis (Kolodkin and Tessier-Lavigne, 2011; Tojima et al., 2011).
∗ Corresponding author. Tel.: +81 52 744 2059. E-mail address: [email protected] (K. Kadomatsu).
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Since axons in the adult mammalian central nervous system (CNS) lack regenerative capacity, it is an important question whether new growth cones generated after injury use the same strategy for regeneration as embryonic neurons employ, and how the injured axons compromise the low intrinsic activity of regeneration (Bradke et al., 2012; Harel and Strittmatter, 2006). Furthermore, the emerging inhibitors after injuries are strong barriers for regeneration. The inhibitors can be categorized into three classes: canonical axon guidance molecules, myelin-derived inhibitors, and chondroitin sulfate proteoglycans (CSPGs) (Giger et al., 2010; Harel and Strittmatter, 2006; Silver and Miller, 2004). Axon guidance molecules include semaphorins, ephrins, netrins and slit. These molecules are expressed upon injury and induce growth cone
0168-0102/$ – see front matter © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. http://dx.doi.org/10.1016/j.neures.2013.10.005
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Fig. 2. Schematic presentation of CS and KS induction and the effects of their ablation. CS and KS are expressed at or around the site of injury. Axonal regeneration/sprouting hardly occur after neuronal injuries. However, if CS or KS are digested, axonal regeneration/sprouting is enhanced and functional recovery is promoted. Fig. 1. Structure of proteoglycan. (A) Structure of proteoglycan. Proteoglycan consists of core protein and glycosaminoglycans (GAGs). GAG is a long sugar chain composed of repeating disaccharide units. (B) Biosynthesis of KS. KS synthesis is accomplished by a cycle of GlcNAc transfer, GlcNAc sulfation and Gal transfer that are mediated by the enzymes 3GnT7, GlcNAc6ST and 4GalT4, respectively. After elongation of the KS chain to some extents, Gal is sulfated by the enzyme KSGal6ST. This oversulfated form of KS is recognized by the antibody 5D4. GlcNAc6ST-1 is responsible for GlcNAc sulfation for the production of 5D4-resposive KS in the brain. Therefore, the deficiency of GlcNAc6ST-1 results in loss of 5D4-resposive KS in the brain.
collapse. Blocking agents against these inhibitors could be beneficial for the functional recovery after injuries, such as spinal cord injury (SCI) (Hata et al., 2006; Kaneko et al., 2006). The myelinderived inhibitors consist of Nogo, myelin-associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein (OMgp). These glycoproteins strongly inhibit neurite outgrowth in vitro. However, the significance of their in vivo functions is still in debate. Thus, triple knockout mice of these three genes have been reported not to exhibit enhanced regeneration of axonal tracts after SCI, while another report showed that the triple knockout mice exhibited greater axonal growth and improved functional recovery (Cafferty et al., 2010; Lee et al., 2010). Silver and colleagues reported in 1990 that axons from the E9 chick dorsal ganglia will not cross a strip of proteoglycans coated on nitrocellulose (Snow et al., 1990a). However, digestion of chondroitin sulfate (CS) abolishes the inhibitory activity of proteoglycans. These findings triggered studies on chondroitin sulfate proteoglycans (CSPGs) as inhibitors of axonal regeneration/sprouting. Glycans consisting of repeating disaccharide units are named glycosaminoglycans (GAGs). CS is a sulfated GAG, the disaccharide units of which are composed of GlcA and GalNAc. CS chains are covalently attached to a core protein, and this whole molecule is generally called CSPG (Fig. 1). It is now accepted that CSPGs act not only as inhibitors of axonal regeneration/sprouting but also as regulators restricting synaptic plasticity and experiencedependent neural plasticity (Bradbury et al., 2002; Frischknecht et al., 2009; Gogolla et al., 2009; Moon et al., 2001; Pizzorusso et al., 2002). We have recently found that the functions of keratan sulfate (KS) and CS merge in the same pathway in inhibition of axonal regeneration/sprouting, and that both sugar chains are essential for the inhibition (Imagama et al., 2011; Ito et al., 2010; Tauchi et al., 2012; Zhang et al., 2006). Here, we overview the currently known biological activities of CSPGs in network rewiring and plasticity after neuronal injuries. Then, we show the functions of KSPGs and discuss the relationship between KS and CS.
2. CSPGs as a major inhibitor of axonal regeneration/sprouting Trauma to the central nervous system (CNS) leads to rupture of the blood–brain barrier, which in turn allows infiltrations of macrophage from the blood and fibroblasts from the meninges, and consequently induces glial scar formation (Silver and Miller, 2004). The glial scar consists of reactive astrocytes and other cellular components as well as extracellular molecules including CSPGs. Round, swollen axonal ends with multiple vacuoles are often found in the glial scar, and are called dystrophic endballs. Dystrophic endballs end in the CSPG deposition in the glial scar, and persist there for at least 9.5–13 weeks after injury (Davies et al., 1999; Li and Raisman, 1995). The formation of dystrophic endballs can be recapitulated in vitro if axons of adult dorsal root ganglion cells are forced to grow against a gradient of CSPGs (Tom et al., 2004). Under this condition, axons cannot extend well. Neurite outgrowth on the substratum evenly coated with CSPGs is also inhibited, and this inhibition is released by the treatment with the CS-degrading enzyme chondroitinase ABC (C-ABC). Consistent with this, C-ABC promotes axonal regeneration after nigrostriatal tract transaction, sprouting of spared fibers in the cuneate nucleus after cervical spinal cord injury (SCI), and functional recovery after SCI (Bradbury et al., 2002; Massey et al., 2006; Moon et al., 2001) (Fig. 2). Therefore, the CS chains of the CSPG moiety seem to be principal actor for the CSPG-mediated inhibition of axonal regeneration/sprouting. 3. CSPGs limit experience-dependent neural plasticity and synaptic plasticity Special extracellular matrix structures, the so-called perineuronal nets (PNNs), are found around a subset of parvalbuminpositive interneurons. PNNs consist of hyaluronan, CSPGs (aggrecan, versican, brevican, neurocan), and tenacin R. Although the precise underlying mechanisms remain elusive, PNNs have been implicated in neural plasticity. For example, ocular dominance plasticity occurs during a postnatal critical period (19–32 postnatal days in mice; 12–36 months in humans), but not in adults. CS digestion with C-ABC restores the ocular dominance plasticity in adult animals (Pizzorusso et al., 2002). Surprisingly, transgenic mice that show an infant-type sulfation pattern of CS also manifest ocular dominance plasticity in adults, suggesting the importance of sulfation modes of CS in PNNs in this plasticity (Miyata et al., 2012). These findings are of clinical importance, since ocular dominance
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plasticity is related to monocular amblyopia. PNNs are also essential for the protection of fear memory from erasure in adult animals. Thus, if the CS in PNNs in the amygdala is digested by C-ABC, fear memory becomes susceptible to erasure. Traumatic memories can be erased by extinction during early development, but PNNs may close this critical period and form erasure-resistant fear memories in adults (Gogolla et al., 2009). Interestingly, other molecules involved in the inhibition of axonal regeneration/sprouting, such as Nogo receptors (NogoRs) and PirB (both are known as receptors for myelin-derived inhibitors), also limit ocular dominance plasticity (McGee et al., 2005; Syken et al., 2006). Furthermore, Nogo receptors restrict spine formation and dendrite branching (Wills et al., 2012). In addition to experience-dependent plasticity, e.g., ocular dominance plasticity and fear memory erasure, CS and hyaluronan have been implicated in synaptic plasticity. Lateral diffusion of glutamate receptors is a critical step for the accumulation of these receptors in the post-synapses, and thus contributes to the synaptic plasticity of excitatory neurons. Digestion of CS or hyaluronan promotes lateral diffusion of glutamate receptors and long term potentiation (Frischknecht et al., 2009). Furthermore, CS is also involved in dendritic spine dynamics. Thus, C-ABC enhances the motility of dendritic spines and induces the appearance of spine head protrusions in a glutamate receptor-independent manner. CSPGs located around dendritic spines are sufficient to restrict spine dynamics independently of PNNs, since microinjection of C-ABC close to dendritic segments can induce spine remodeling (Orlando et al., 2012).
4. Receptors for CSPGs Several receptors for CS have been postulated. These include PTP, LAR, NogoR1, NogoR3, and contactin-1 (Coles et al., 2011; Dickendesher et al., 2012; Fisher et al., 2011; Mikami et al., 2009; Shen et al., 2009). Blocking or ablation of PTP, LAR, NogoR1 and NogoR3 restores the neurite outgrowth inhibited by CSPGs and promotes axonal regeneration/sprouting in vivo. Contactin-1 binds to CS-E (E indicates a type of sulfation), and is involved in CS-Emediated neurite outgrowth under some conditions (Mikami et al., 2009).
Regarding the mechanisms of CS action, it is worthy of note that CSPGs and heparan sulfate proteoglycans (HSPGs) show distinct localization and functions. HSPGs are located on the surface of axons, while CSPGs are rich in extracellular spaces. Structural analysis of PTP has revealed that HSPGs bind to PTP and promote oligomerization of PTP on axons (Coles et al., 2011). Once the production of CSPGs in the extracellular space is enhanced after injuries, CSPGs bind to PTP. As a consequence, the PTP oligomers may be disrupted and PTP may be turned on. In this way, HSPGs and CSPGs work as a switch in response to injury. The transmembrane protein semaphorin 5A (Sema5A) is also present at the intersection between HSPGs and CSPGs. The extension of axons of the fasciculus retroflexus is promoted by Sema5A through binding of HSPGs on the axons, while CSPGs precisely localized in the extracellular space convert Sema5A from an attractive to an inhibitory guidance cue (Kantor et al., 2004).
5. KSPGs as a major inhibitor of axonal regeneration/sprouting KS is expressed in the glial roof plate in the spinal cord during E13.5-15.5 in a temporal- and spatial-specific manner (Snow et al., 1990b). The commissural and dorsal column axons never cross the roof plate, suggesting that KS functions as a guidance cue for these axons. As described above, axons from the dorsal root ganglia will not cross a strip of proteoglycans coated on nitrocellulose. This inhibition is abolished by digestion of not only CS but also KS (Snow et al., 1990a). Furthermore, mossy fibers originate from granule cells of the dentate gyrus and synapse on the proximal apical dendrites of CA pyramidal neurons within a narrow band called the stratum lucidum. Treatment with keratanase, but not C-ABC, leads to the misrouting of mossy fibers in rat hippocampal slice cultures (Butler et al., 2004). Interestingly, mice deficient in PTP, a receptor for CS, show a similar phenotype, o.e., increased collateral branching of mossy fiber, suggesting that PTP might work as a KS receptor (Horn et al., 2012). These findings suggest that KS may also be vital for axonal guidance and/or axonal regeneration/sprouting. In line with this, KS expression is upregulated at SCI lesions (Imagama et al., 2011; Ito et al., 2010; Jones and Tuszynski, 2002) (Fig. 2).
Fig. 3. KS expression and in vivo neurite growth after a cortical stab wound. CS expression is induced at the site of a stab wound in both wild-type and GlcNAc6ST-1 KO mice. In contrast, KS expression is only induced in wild-type mice, but not in GlcNAc6ST-1 KO mice. As a result, neurite outgrowth in vivo that is represented by phosphoneurofilament staining is enhanced in GlcNAc6ST-1 KO mice. These data suggest that KS is an essential inhibitor of axonal regeneration/sprouting after neuronal injuries. Modified from Zhang et al. (2006).
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Fig. 4. Effects of KS and CS ablation on neurite growth in vitro and on functional recovery after spinal cord injury. In vitro neurite outgrowth assay indicates that proteoglycan (PG) inhibits neurite outgrowth while CS-digestion (C-ABC) or KS-digestion (K-II) reverses this inhibition. Interestingly, the combination of CS- and KS-digestion shows neither additive nor synergistic effect. Consistent with these data, CS- or KS-digestion promotes motor function recovery after SCI. But, the combination does not show additive or synergistic effect. Modified from Imagama et al. (2011).
KS is made up of repeating disaccharide units consisting of Gal and GlcNAc. For the generation of a KS chain, GlcNAc is sulfated at the C6 position, Gal is transferred and GlcNAc is further transferred to the chains. Therefore, GlcNAc sulfation is essential for the KS chain elongation (Fig. 1). This sulfation is mediated by GlcNAc6ST (GlcNAc 6-O-sulfotransferase). Four and five GlcNAc6STs have been identified in mice and humans, respectively, and are implicated in various biological activities (Uchimura and Rosen, 2006). Among them, we cloned mouse GlcNAc6ST-1 and generated its deficient mice (Uchimura et al., 1998, 2004). A developmentally regulated expression of KS can be determined by the oversulfated KS-specific antibody 5D4, but this expression is completely abolished in GlcNAc6ST-1 KO mice (Zhang et al., 2006). While wild-type mice show induction of KS and CS expression at stab wound sites in the cerebral cortex, GlcNAc6ST-1 KO mice exhibit CS induction, but not KS, in response to this trauma. To our surprise, GlcNAc6ST-1 KO mice show the enhanced neurite outgrowth in vivo at the stab wound site even if CS expression is induced there. These data suggest that KS inhibits neurite growth independently of CS (Zhang et al., 2006) (Fig. 3). GlcNAc6ST-1 KO mice also show significantly better functional recovery as well as axonal regeneration/sprouting after SCI (Ito et al., 2010). Consistent with this, chick brain-derived proteoglycans that are commonly used as representative proteoglycans to inhibit neurite growth in vitro contain not only CSPGs but also KSPGs (Imagama et al., 2011). KS digestion of chick brain-derived proteoglycans reverses the inhibition of neurite growth in vitro. Local administration of keratanase II, an enzyme that specifically digests KS, to the injured site promotes the recovery of motor and sensory functions after SCI (Imagama et al., 2011). 6. KS and CS work in the same pathway in the inhibition of axonal regeneration/sprouting KS digestion and CS digestion show comparable effects on functional recovery in vivo and neurite growth in vitro, but the combination of these digestions does not exert additive or synergistic effects (Fig. 4). These findings provide new insights into the mechanisms of proteoglycan-mediated inhibition of axonal regeneration/sprouting. Both KS and CS are essential for this inhibition. We speculate that KS and CS might share a complex of receptors, each of which individually binds CS or KS. Alternatively, the receptors might exchange cross-talk to mediate the same common intracellular signaling pathway. Considering that CSPGs are also
involved in neural plasticity, the elucidation of KS and CS signaling would have a major impact on our understanding of neuronal network rewiring and plasticity after injury and in treating disorders such as SCI, stroke and neurodegenerative diseases. However, in order to uncover the biological significance of KS and CS, an integrative and comprehensive understanding of the mechanisms underlying network rewiring and plasticity after neuronal injuries may be necessary. For example, unlike adult CNS neurons, neural stem cell-derived neurons extend axons across the lesion and over long distances. These neurons form electrophysiological relays across the lesion, which leads to functional recovery. Human neural stem cell lines show similar activities (Lu et al., 2012). Another report had demonstrated that the combination of a peripheral nerve autograft and C-ABC promotes the plasticity of spared tracts and long-distance regeneration of essential pathways, leading to a remarkable recovery of diaphragmatic function (Alilain et al., 2011). Furthermore, C-ABC in combination with rehabilitation has been shown to promote functional recovery in acute SCI as well as chronic SCI (Garcia-Alias et al., 2009; Wang et al., 2011). It has also been reported that cells expressing the CSPG NG2 stabilize dystrophic endballs rather than inhibiting regenerating sensory axons in the inhibitory environment of the glial scar (Busch et al., 2010). The continued clarification of these issues, i.e., the distinct intrinsic regeneration potentials of neurons, the cross-talk between proteoglycans and rehabilitation, and the formation of dystrophic endballs, should help to promote studies on KS and CS. Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific Research on Innovative Areas (No. 23110002 to K.K.) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan, Grant-in-Aid for Research Activity Start-up (No. 24890084 to K.S.), and by funds from the Global COE program, MEXT, to Nagoya University. References Alilain, W.J., Horn, K.P., Hu, H., Dick, T.E., Silver, J., 2011. Functional regeneration of respiratory pathways after spinal cord injury. Nature 475, 196–200. Bradbury, E.J., Moon, L.D., Popat, R.J., King, V.R., Bennett, G.S., Patel, P.N., Fawcett, J.W., McMahon, S.B., 2002. Chondroitinase ABC promotes functional recovery after spinal cord injury. Nature 416, 636–640. Bradke, F., Fawcett, J.W., Spira, M.E., 2012. Assembly of a new growth cone after axotomy: the precursor to axon regeneration. Nature Reviews. Neuroscience 13, 183–193.
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