Gene delivery to overcome astrocyte inhibition of axonal growth: An in vitro Model of the glial scar

After injury to the central nervous system, a glial scar develops that physically and biochemically inhibits axon growth. In the scar, activated astrocytes secrete inhibitory extracellular matrix, of which chondroitin sulfate proteoglycans (CSPGs) are considered the major inhibitory component. An inhibitory interface of CSPGs forms around the lesion and prevents axons from traversing the injury, and decreasing CSPGs can enhance axon growth. In this report, we established an in vitro interface model of activated astrocytes and subsequently investigated gene delivery as a means to reduce CSPG levels and enhance axon growth. In the model, a continuous interface of CSPG producing astrocytes was created with neurons seeded opposite the astrocytes, and neurite crossing, stopping, and turning were evaluated as they approached the interface. We investigated the efficacy of lentiviral delivery to degrade or prevent the synthesis of CSPGs, thereby removing CSPG inhibition of neurite growth. Lentiviral delivery of RNAi targeting two key CSPG synthesis enzymes, chondroitin polymerizing factor and chondroitin synthase‐1, decreased CSPGs, and reduced inhibition by the interface. Degradation of CSPGs by lentiviral delivery of chondroitinase also resulted in less inhibition and more neurites crossing the interface. These results indicate that the interface model provides a tool to investigate interventions that reduce inhibition by CSPGs, and that gene delivery can be effective in promoting neurite growth across an interface of CSPG producing astrocytes. Biotechnol. Bioeng. 2013; 110: 947–957. © 2012 Wiley Periodicals, Inc.     

Easy HPLC-based separation and quantitation of chondroitin sulphate and hyaluronan disaccharides after chondroitinase ABC treatment

The sulphation patterns of glycosaminoglycan (GAG) chains are decisive for the biological activity of their proteoglycan (PG) templates for sugar chain polymerization and sulphation. The amounts and positions of sulphate groups are often determined by HPLC analysis of disaccharides resulting from enzymatic degradation of the GAG chains. While heparan sulphate (HS) and heparin are specifically degraded by heparitinases, chondroitinases not only degrade chondroitin sulphate (CS) and dermatan sulphate (DS), but also the protein-free and unsulphated GAG hyaluronan (HA). Thus, disaccharide preparations derived by chondroitinase degradation may be contaminated by HA disaccharides. The latter will often comigrate in HPLC chromatograms with unsulphated disaccharides derived from CS. We have investigated how variation of pH, amount of enzyme, and incubation time affects disaccharide formation from CS and HA GAG chains. This allowed us to establish conditions where chondroitinase degrades CS completely for quantification of all the resulting disaccharides, with negligible degradation of HA, allowing subsequent HA analysis. In addition, we present simple methodology for disaccharide analysis of small amounts of CS attached to a hybrid PG carrying mostly HS after immune isolation. Both methods are applicable to small amounts of GAGs synthesized by polarized epithelial cells cultured on permeable supports.     

Chondroitin sulfate proteoglycans in neural development and regeneration

Proteoglycans are of two main types, chondroitin sulfate (CSPGs) and heparin sulfate (HSPGs). The CSPGs act mainly as barrier-forming molecules, whereas the HSPGs stabilise the interactions of receptors and ligands. During development CSPGs pattern cell migration, axon growth pathways and axon terminations. Later in development and in adulthood CSPGs associate with some classes of neuron and control plasticity. After damage to the nervous system, CSPGs are the major axon growth inhibitory component of the glial scar tissue that blocks successful regeneration. CSPGs have a variety of roles in the nervous system, including binding to molecules and blocking their action, presenting molecules to cells and axons, localising active molecules to particular sites and presenting growth factors to their receptors.     

Chondroitinase ABC has a long-lasting effect on chondroitin sulphate glycosaminoglycan content in the injured rat brain

Chondroitin sulphate proteoglycans (CSPGs) are axon growth inhibitory molecules present in the glial scar that play a part in regeneration failure after damage to the CNS and which restrict CNS plasticity. Removal of chondroitin sulphate glycosaminoglycan (GAG) chains with chondroitinase-ABC (chABC) in models of CNS injury promotes both axon regeneration and plasticity. We have analysed the immediate and long-term effects of a single injection of chABC on CSPGs, GAGs and axon regeneration. We made unilateral nigrostriatal lesions in adult rats accompanied by an adjacent infusion of either chABC or a bacterial-derived control enzyme (penicillinase). Within 24 h of chABC treatment there was digestion of GAGs, including hyaluronan, and a reduction in neurocan in an area extending 1.5 mm around the injection site. Around 50% of GAG is inaccessible to chABC digestion, even in tissue digested in vitro, which probably represents intracellular stores. In control penicillinase treated animals, total GAGs recovered from the lesioned brains were up-regulated by 4-fold 7 days after injury and gradually decreased to normal at 28 days post-lesion. In chondroitinase-treated animals, the total GAG remained at low level throughout the 28-day experimental period. This suggests the persistence of active chABC for at least 10 days after injection which is able to digest CSPGs released from cells during this time. This was confirmed by immunological detection of enzyme for 10 days and by retrieval of active enzyme from the brain at 10 days after injection. Our results suggest that a single injection of chABC can produce an environment conducive to CNS repair for over 10 days.     

The low sulfated chondroitin sulfate proteoglycans of human placenta have sulfate group-clustered domains that can efficiently bind Plasmodium falciparum-infected erythrocytes

Plasmodium falciparum infection in pregnant women results in the chondroitin 4-sulfate-mediated adherence of the parasite-infected red blood cells (IRBCs) in the placenta, adversely affecting the health of the fetus and mother. We have previously shown that unusually low sulfated chondroitin sulfate proteoglycans (CSPGs) in the intervillous spaces of the placenta are the receptors for IRBC adhesion, which involves a chondroitin 4-sulfate motif consisting of six disaccharide moieties with approximately 30% 4-sulfated residues. However, it was puzzling how the placental CSPGs, which have only approximately 8% of the disaccharide 4-sulfated, could efficiently bind IRBCs. Thus, we undertook to determine the precise structural features of the CS chains of placental CSPGs that interact with IRBCs. We show that the placental CSPGs are a mixture of two major populations, which are similar by all criteria except differing in their sulfate contents; 2-3% and 9-14% of the disaccharide units of the CS chains are 4-sulfated, and the remainder are nonsulfated. The majority of the sulfate groups in the CSPGs are clustered in CS chain domains consisting of 6-14 repeating disaccharide units. While the sulfate-rich regions of the CS chains contain 20-28% 4-sulfated disaccharides, the other regions have little or no sulfate. Further, we find that the placental CSPGs are able to efficiently bind IRBCs due to the presence of 4-sulfated disaccharide clusters. The oligosaccharides corresponding to the sulfate-rich domains of the CS chains efficiently inhibited IRBC adhesion. Thus, our data demonstrate, for the first time, the unique distribution of sulfate groups in the CS chains of placental CSPGs and that these sulfate-clustered domains have the necessary structural elements for the efficient adhesion of IRBCs, although the CS chains have an overall low degree of sulfation.     

Peripheral nervous system genes expressed in central neurons induce growth on inhibitory substrates

Trauma to the spinal cord and brain can result in irreparable loss of function. This failure of recovery is in part due to inhibition of axon regeneration by myelin and chondroitin sulfate proteoglycans (CSPGs). Peripheral nervous system (PNS) neurons exhibit increased regenerative ability compared to central nervous system neurons, even in the presence of inhibitory environments. Previously, we identified over a thousand genes differentially expressed in PNS neurons relative to CNS neurons. These genes represent intrinsic differences that may account for the PNS's enhanced regenerative ability. Cerebellar neurons were transfected with cDNAs for each of these PNS genes to assess their ability to enhance neurite growth on inhibitory (CSPG) or permissive (laminin) substrates. Using high content analysis, we evaluated the phenotypic profile of each neuron to extract meaningful data for over 1100 genes. Several known growth associated proteins potentiated neurite growth on laminin. Most interestingly, novel genes were identified that promoted neurite growth on CSPGs (GPX3, EIF2B5, RBMX). Bioinformatic approaches also uncovered a number of novel gene families that altered neurite growth of CNS neurons.     

Chondroitin sulfate proteoglycans of bovine cornea: structural characterization and assessment for the adherence of Plasmodium falciparum-infected erythrocytes

The structures of the bovine corneal chondroitin sulfate (CS) chains and the nature of core proteins to which these chains are attached have not been studied in detail. In this study, we show that structurally diverse CS chains are present in bovine cornea and that they are mainly linked to decorin core protein. DEAE-Sephacel chromatography fractionated the corneal chondroitin sulfate proteoglycans (CSPGs) into three distinct fractions, CSPG-I, CSPG-II, and CSPG-III. These CSPGs markedly differ in their CS and dermatan sulfate (DS) contents, and in particular the CS structure-the overall sulfate content and 4- to 6-sulfate ratio. In general, the CS chains of the corneal CSPGs have low to moderate levels (15-64%) of sulfated disaccharides and 0-30% DS content. Structural analysis indicated that the DS disaccharide units in the CS chains are segregated as large blocks. We have also assessed the suitability of the corneal CSPGs as an alternative to placental CSPG or the widely used bovine tracheal chondroitin sulfate A (CSA) for studying the structural interactions involved in the adherence of Plasmodium falciparum-infected red blood cells (IRBCs) to chondroitin 4-sulfate. The data demonstrate that the corneal CSPGs efficiently bind IRBCs, and that the binding strength is either comparable or significantly higher than the placental CSPG. In contrast, the IRBC binding strength of bovine tracheal CSA is markedly lower than the human placental and bovine corneal CSPGs. Thus, our data demonstrate that the bovine corneal CSPG but not tracheal CSA is suitable for studying structural interactions involved in IRBC-C4S binding.     

6-Sulphated chondroitins have a positive influence on axonal regeneration

Chondroitin sulphate proteoglycans (CSPGs) upregulated in the glial scar inhibit axon regeneration via their sulphated glycosaminoglycans (GAGs). Chondroitin 6-sulphotransferase-1 (C6ST-1) is upregulated after injury leading to an increase in 6-sulphated GAG. In this study, we ask if this increase in 6-sulphated GAG is responsible for the increased inhibition within the glial scar, or whether it represents a partial reversion to the permissive embryonic state dominated by 6-sulphated glycosaminoglycans (GAGs). Using C6ST-1 knockout mice (KO), we studied post-injury changes in chondroitin sulphotransferase (CSST) expression and the effect of chondroitin 6-sulphates on both central and peripheral axon regeneration. After CNS injury, wild-type animals (WT) showed an increase in mRNA for C6ST-1, C6ST-2 and C4ST-1, but KO did not upregulate any CSSTs. After PNS injury, while WT upregulated C6ST-1, KO showed an upregulation of C6ST-2. We examined regeneration of nigrostriatal axons, which demonstrate mild spontaneous axon regeneration in the WT. KO showed many fewer regenerating axons and more axonal retraction than WT. However, in the PNS, repair of the median and ulnar nerves led to similar and normal levels of axon regeneration in both WT and KO. Functional tests on plasticity after the repair also showed no evidence of enhanced plasticity in the KO. Our results suggest that the upregulation of 6-sulphated GAG after injury makes the extracellular matrix more permissive for axon regeneration, and that the balance of different CSs in the microenvironment around the lesion site is an important factor in determining the outcome of nervous system injury.     

硫酸软骨素蛋白多糖与神经系统的发育和再生

硫酸软骨素蛋白多糖（chondroitin sulfate proteoglycans,CSPGs）是中枢神经系统（CNS）细胞外基质中的重要组成成分,在CNS的发育、成熟后正常功能的维持中发挥重要功能,如发育中影响神经细胞的迁移和轴突生长,成年后参与神经可塑性的控制等;而病理条件下,如CNS受损后又可做为胶质瘢痕的重要组分抑制受损神经的再生。研究发现,用酶降解CSPGs的糖氨多糖链或阻断其合成可以有效地削弱CSPGs对受损神经的抑制作用,促进轴突再生。然而,精确调控CSPGs特定时空表达模式的分子机制,以及功能发挥所涉及的完整信号转导通路还有待进一步研究。     

Integrins and the extracellular matrix: key mediators of development and regeneration of the sensory nervous system

The somatosensory nervous system is responsible for the transmission of a multitude of sensory information from specialized receptors in the periphery to the central nervous system. Sensory afferents can potentially be damaged at several sites: in the peripheral nerve; the dorsal root; or the dorsal columns of the spinal cord; and the success of regeneration depends on the site of injury. The regeneration of peripheral nerve branches following injury is relatively successful compared to central branches. This is largely attributed to the presence of neurotrophic factors and a Schwann cell basement membrane rich in permissive extracellular matrix (ECM) components which promote axonal regeneration in the peripheral nerve. Modulation of the ECM environment and/or neuronal integrins may enhance regenerative potential of sensory neurons following peripheral or central nerve injury or disease. This review describes the interactions between integrins and ECM molecules (particularly the growth supportive ligands, laminin, and fibronectin; and the growth inhibitory chondroitin sulfate proteoglycans (CSPGs)) during development and regeneration of sensory neurons following physical injury or neuropathy.     

Altered content composition and structure of glycosaminoglycans and proteoglycans in gastric carcinoma

Glycosaminoglycans (GAGs) in proteoglycan (PG) forms or as free GAGs are implicated in the growth and progression of malignant tumors. These macromolecules were investigated in human gastric carcinoma (HGC) and compared with those in human normal gastric mucosa (HNG). We report that HGC contained about 2-fold increased amounts of GAGs in comparison to HNG. Specifically, HGC showed 3- and 2.5-fold net increase in chondroitin sulphate (CS) and hyaluronan (HA) contents, respectively. Dermatan sulphate (DS) was slightly increased, but the amount of heparan sulphate (HS) was decreased. Of particular, interest were the quite different sulphation profiles of CS and DS chains in HGC in which, non-sulphated and 6-sulphated disaccharide units were increased 10 and 4 times, respectively, in comparison to HNG. On PG level, three different populations were identified in both HNG and HGC, being HSPGs, versican (CS/DS chains) and decorin (CS/DS chains). In HGC, the amounts of versican and decorin were significantly increased about 3- and 8-fold, respectively. These PGs were also characterized by marked decrease in hydrodynamic size and GAG content per PG molecule. Analysis of Delta-disaccharide of versican and decorin from HGC showed an increase of 6-sulphated Delta-disaccharides (Delta di-6S) and non-sulphated Delta-disaccharides (Delta di-0S) with a parallel decrease of 4-sulphated Delta-disaccharides (Delta di-4S) as compared to HNG, which closely correlated with the increase of CS content. In addition, the accumulation of core proteins of versican and decorin in HGC was also associated with many post-translational modifications, referring to the number, size, degree and patterns of sulphation and epimerization of CS/DS chains. Studies on the modified metabolism of PGs/GAGs are under progress and will help in deeper understanding of the environment in which tumor cells proliferate and invade.     

Synthesis of glycosaminoglycans by cloned bovine endothelial cells cultured on collagen gels

Sulphated glycosaminoglycans have been analysed in cloned bovine aortic endothelial cells cultured on collagen gels after incubation with [3H]glucosamine and Na2(35)SO4. Radioactive products were analysed in the culture medium, in sequential collagenase and trypsin extracts of the cell monolayer and the associated extracellular matrix, and in the remaining viable cells. Heparan sulphate and chondroitin sulphate were found in each compartment: the heparan sulphate had a low degree of sulphation (approximately 0.4 N-sulphate and 0.2 O-sulphate groups per disaccharide unit on average). In the nitrous acid scission products of heparan sulphate, O-sulphated substituents were confined to disaccharide and tetrasaccharide fragments, indicating that local regions of the chain (which might be susceptible to excission by the platelet endoglycosidase) are highly sulphated. Only minor structural differences in heparan sulphate were observed between the various compartments. In contrast the chondroitin sulphate found in the collagenase extract had a higher iduronic acid content than corresponding material in the trypsin extract and the culture medium, indicating that collagenase and trypsin may extract glycosaminoglycans from different regions of the extracellular and pericellular matrix.     

Chondroitin/dermatan sulfate in the central nervous system

In the central nervous system (CNS) chondroitin sulfate proteoglycans, as one of the major barrier-forming molecules, influence cell migration patterns and axon pathfinding. By contrast, chondroitin sulfate side chains often form hybrid chains with dermatan sulfate and serve as a neural stem cell marker and neurogenic/neuritogenic molecules involved in neural stem cell proliferation. Hybrid chondroitin/dermatan sulfate chains are also involved in formation of the neural network by capturing and presenting heparin-binding growth factors like basic fibroblast growth factor, pleiotrophin, and hepatocyte growth factor to stem cells or neuronal cells. Research tools for structural glycobiology are emerging to perform a high-throughput screening of glycosaminoglycans for the binding to ligands, to decipher sulfation patterns of rare functional oligosaccharide sequences and to build structural models for the shape of such sulfated oligosaccharides.     

Sugar codes for axons?

Proteoglycans are complex macromolecules with the potential for extraordinary diversity. Several recent studies have demonstrated important roles for heparan sulfate and chondroitin sulfate proteoglycans (HSPGs and CSPGs) in axon pathfinding and have linked HSPGs to specific signaling pathways. More speculatively, there are hints of a "sugar code," in which specific sugar modifications might act instructively in guidance decisions. This raises the intriguing possibility that the complexity of neuronal wiring may in part reflect the complexity of proteoglycan modifications.     

Chondroitin sulfate: a key molecule in the brain matrix

Chondroitin sulfate is a glycosaminoglycan composed of N-acetylgalactosamine and glucuronic acid. It attaches to a core protein to form chondroitin sulfate proteoglycan (CSPG). Being a major component of the brain extracellular matrix, CSPGs are involved in neural development, axon pathfinding and guidance, plasticity and also regeneration after injury in the nervous system. In this review, we shall discuss the structure, the biosynthetic pathway, its functions in the nervous system and how we can improve regeneration in the nervous system by modulating its structure and binding properties.     

Developmental distribution of glycosaminoglycans in embryonic rat brain: Relationship to axonal tract formation

Glycosaminoglycans, the sugar moieties of proteoglycans, modulate axonal growth in vitro. However, their anatomical distribution in relation to developing axonal tracts in the rat brain has not been studied. Here, we examined the immunohistochemical distribution of chondroitin-6-sulfate and chondroitin-4-sulfate, two related glycosaminoglycan epitopes, which are present in three types of glycosaminoglycans: chondroitin sulfate C, chondroitin sulfate A, and chondroitin sulfate B. Further, we compared their distribution pattern to that of axonal tract development. Both glycosaminoglycan epitopes showed a heterogeneous spatiotemporal distribution within the developing rat brain. However, the expression of chondroitin-4-sulfate was more restricted than that of chondroitin-6-sulfate, although both epitopes were detected from embryonic day 13 until the day of birth, overlapping in many regions of the central nervous system including cortex, hippocampus, thalamus, and hindbrain. After birth, the levels of expression of both glycosaminoglycan epitopes progressively decreased and were practically undetectable after the first postnatal week. The expression of chondroitin-6-sulfate and, to a lesser extent, that of chondroitin-4-sulfate, was preferentially associated to the extracellular matrix surrounding specific axon bundles. However, the converse association was not true, and several apparently similar types of axon developed on a substrate devoid of both types of glycosaminoglycan epitopes. These results provide an anatomical background for the idea that different types of glycosaminoglycans may contribute to establish the complex set of guidance cues necessary for the specific development of defined axon tracts in the central nervous system. © 1996 John Wiley & Sons, Inc.     

Neuronal glycosylation differentials in normal, injured and chondroitinase-treated environments

Glycosylation is found ubiquitously throughout the central nervous system (CNS). Chondroitin sulphate proteoglycans (CSPGs) are a group of molecules heavily substituted with glycosaminoglycans (GAGs) and are found in the extracellular matrix (ECM) and cell surfaces. Upon CNS injury, a glial scar is formed, which is inhibitory for axon regeneration. Several CSPGs are up-regulated within the glial scar, including NG2, and these CSPGs are key inhibitory molecules of axonal regeneration. Treatment with chondroitinase ABC (ChABC) can neutralise the inhibitory nature of NG2. A gene expression dataset was mined in silico to verify differentially regulated glycosylation-related genes in neurons after spinal cord injury and identify potential targets for further investigation. To establish the glycosylation differential of neurons that grow in a healthy, inhibitory and ChABC-treated environment, we established an indirect co-culture system where PC12 neurons were grown with primary astrocytes, Neu7 astrocytes (which overexpress NG2) and Neu7 astrocytes treated with ChABC. After 1, 4 and 8 days culture, lectin cytochemistry of the neurons was performed using five fluorescently-labelled lectins (ECA MAA, PNA, SNA-I and WFA). Usually α-(2,6)-linked sialylation scarcely occurs in the CNS but this motif was observed on the neurons in the injured environment only at day 8. Treatment with ChABC was successful in returning neuronal glycosylation to normal conditions at all timepoints for MAA, PNA and SNA-I staining, and by day 8 in the case of WFA. This study demonstrated neuronal cell surface glycosylation changes in an inhibitory environment and indicated a return to normal glycosylation after treatment with ChABC, which may be promising for identifying potential therapies for neuronal regeneration strategies.     

The inhibitory effects of chondroitin sulfate proteoglycans on oligodendrocytes

The formation of the glial scar following a spinal cord injury presents a significant barrier to the regenerative process. It is primarily composed of chondroitin sulfate proteoglycans (CSPGs) that can inhibit axonal sprouting and regeneration. Although the inhibitory effects on neurons are well documented, little is known about their effects on oligodendrocyte progenitor cells (OPCs). In this study, we examined the effects of CSPGs on OPC process outgrowth and differentiation in vitro. The results show that specific CSPGs, in particularly those highly up-regulated following spinal cord injury, inhibit OPC process outgrowth and differentiation, and that treatment with chondroitinase ABC can completely reverse this inhibition. Additionally, treatment with the Rho kinase inhibitor Y-27632 also reverses the observed inhibition, implicating the activation of Rho kinase in the CSPG inhibition of OPC growth. Taken together, these findings demonstrate that the CSPGs found within the glial scar are not only inhibitory to neurons, but also to OPCs. Moreover, this study shows that chondroitinase ABC treatment, having shown promise in promoting axonal regeneration, may also enhance remyelination.     

Central Nervous System Regeneration Inhibitors and their Intracellular Substrates

Injury to the central nervous system (CNS) initiates a cascade of responses that is inhibitory to the regeneration of neurons and full recovery. At the site of injury, glial cells conspire with an inhibitory biochemical milieu to construct both physical and chemical barriers that prevent the outgrowth of axons to or beyond the lesion site. These inhibitors include factors derived from myelin, repulsive guidance cues, and chondroitin sulfate proteoglycans. Each bind receptors on the axon surface to initiating intracellular signaling cascades that ultimately result in cytoskeletal reorganization and growth cone collapse. Here, we present an overview of the molecules, receptors, and signaling pathways that inhibit CNS regeneration, with a particular focus on the intracellular signaling machinery that may function as convergent targets for multiple inhibitory ligands.     

Constant and variable domains of different disaccharide structure in corneal keratan sulphate chains.

Four peptidokeratan sulphate fractions of different Mr and degree of sulphation were cut from the pig corneal keratan sulphate distribution spectrum. After exhaustive digestion with keratanase, the fragments were separated on DEAE-Sephacel and Bio-Gel P-10 and analysed for their Mr, degree of sulphation and amino sugar and neutral sugar content. It was found that every glycosaminoglycan chain is constructed of a constant domain of non-sulphated and monosulphated disaccharide units and a variable domain of disulphated disaccharide units. Total neuraminic acid of the four peptidokeratan sulphates was recovered from their isolated linkage-region oligosaccharides. In kinetic studies, the four peptidokeratan sulphates were investigated for Mr distribution after various incubation times with keratanase. There was a continuous shift towards lower Mr and no appearance of a distinct intermediate-sized product at any degradation time. The linkage-region oligosaccharide was already being liberated after a very short incubation period. From the results of these kinetic investigations in connection with the results of neuraminic acid analyses it is suggested that there exists only one disaccharide chain per peptidokeratan sulphate molecule. A model of corneal keratan sulphate is postulated. One of the alpha-mannose residues in the linkage region is bound to an oligosaccharide consisting of a lactosamine and a terminal sialic acid. The other alpha-mannose residue is attached to the disaccharide chain. This chain contains one or two non-sulphated disaccharide units at the reducing end, followed by 10-12 monosulphated disaccharide units. The disulphated disaccharide moiety of variable length is positioned at the non-reducing end of the chain.