Chondroitin sulphate proteoglycans in the central nervous system: changes and synthesis after injury

Chondroitin sulphate proteoglycans (CSPGs) are up-regulated in the central nervous system after injury, specifically around the lesion site where the glial scar forms. This structure contains astrocytes, oligodendrocyte precursor cells, microglia and meningeal cells, and forms an inhibitory substrate for axon re-growth. CSPGs have been shown to be closely involved in this neuronal growth inhibition, specifically through their sugar chains. These chains are composed of repeats of the same disaccharide unit carrying sulphate groups in different positions. The sulphation pattern directly influences the CSPG binding properties and function; the specific sulphation pattern required for the inhibitory activity of these molecules on axon growth is unknown at present. The expression of the chondroitin sulphotransferases, which sulphate the disaccharide residues of CSPGs and thus are responsible for the structural diversity of the chondroitin sulphate sugar chains, is regulated differently in central nervous system during development and after injury, suggesting the implication of a specific sulphation pattern in the inhibitory activity of CSPGs.     

Smad proteins differentially regulate transforming growth factor-β-mediated induction of chondroitin sulfate proteoglycans

Traumatic injury to the CNS results in increased expression and deposition of chondroitin sulfate proteoglycans (CSPGs) that are inhibitory to axonal regeneration. Transforming growth factor-β (TGF-β) has been implicated as a major mediator of these changes, but the mechanisms through which TGF-β regulates CSPG expression are not known. Using lentiviral expressed Smad-specific ShRNA we show that TGF-β induction of CSPG expression in astrocytes is Smad-dependent. However, we find a differential dependence of the synthetic machinery on Smad2 and/or Smad3. TGF-β induction of neurocan and xylosyl transferase 1 required both Smad2 and Smad3, whereas induction of phosphacan and chondroitin synthase 1 required Smad2 but not Smad3. Smad3 knockdown selectively reduced induction of chondroitin-4-sulfotransferase 1 and the amount of 4-sulfated CSPGs secreted by astrocytes. Additionally, Smad3 knockdown in astrocytes was more efficacious in promoting neurite outgrowth of neurons cultured on the TGF-β-treated astrocytes. Our data implicate TGF-β Smad3-mediated induction of 4-sulfation as a critical determinant of the permissiveness of astrocyte secreted CSPGs for axonal growth.     

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.     

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.     

X-irradiation reduces lesion scarring at the contusion site of adult rat spinal cord

Spinal cord injury (SCI) results in cell death and tissue destruction, and ultimately cavitation followed by the formation of lesion scars at the injury site. The lesion scars include an astrocytic component (glial scar) and a fibroblastic component (connective tissue scar). The purpose of the present study is to determine if X-irradiation could minimize the formation of lesion scars and reduce the levels of chondroitin sulfate proteoglycans (CSPGs) in the contusion SCI model of the adult rat. Two weeks after SCI, a connective tissue scar formed at the injury site consisting primarily of fibroblasts and exhibits strong CSPG immunoreactivity. The fibroblasts might originate from the connective tissue of pia mater or arachnoid mater. At the same time, reactive astrocytes in the spared tissue accumulate surrounding the lesion cavity to form a thick glial scar with significant enhancement of glial fibrillary acidic protein (GFAP) and CSPG immunoreactivity. After X-irradiation (40 Gy) of the injury site 2 days post-injury, that results in an attenuated dose to the lesion, the connective tissue scar was not observed, and accordingly, almost no CSPG immunoreactivity was detected at this area. Meanwhile, the glial scar and its CSPG immunoreactivity were prominently reduced. X-irradiation did not show significant improvement in locomotor recovery, but resulted in a slight delay of body weight recovery following injury. This preparative treatment could be used to reduce secondary scarring in the lesion resulting in an enriched site for further treatment such as growth related transplantation.     

Antisense vimentin cDNA combined with chondroitinase ABC reduces glial scar and cystic cavity formation following spinal cord injury in rats

The formation of glial scar and cystic cavities restricts axon regeneration after spinal cord injury. Chondroitin sulphate proteoglycans (CSPGs) are regarded as the prominent inhibitory molecules in the glial scar, and their inhibitory effects may be abolished in part by chondroitinase ABC (ChABC), which can digest CSPGs. CSPGs are secreted mostly by reactive astrocytes, which form dense scar tissues. The intermediate filament protein vimentin underpins the cytoskeleton of reactive astrocytes. Previously we have shown that retroviruses carrying full-length antisense vimentin cDNA reduce reactive gliosis. Here we administered both antisense vimentin cDNA and ChABC to hemisected rat spinal cords. Using RT-PCR, Western blotting and immunohistochemistry, we found that the combined treatment reduced the formation of glial scar and cystic cavities through degrading CSPGs molecules and inhibiting intermediate filament proteins. The modified intra- and extra-cellular architecture may alter the physical and biochemical characteristics of the scar, and the combined therapy might be used to inhibit glial scar formation.     

GM-CSF reduces expression of chondroitin sulfate proteoglycan (CSPG) core proteins in TGF-β-treated primary astrocytes

GM-CSF plays a role in the nervous system, particularly in cases of injury. A therapeutic effect of GM-CSF has been reported in rat models of various central nervous system injuries. We previously showed that GM-CSF could enhance long-term recovery in a rat spinal cord injury model, inhibiting glial scar formation and increasing the integrity of axonal structure. Here, we investigated molecular the mechanism(s) by which GM-CSF suppressed glial scar formation in an in vitro system using primary astrocytes treated with TGF-β. GM-CSF repressed the expression of chondroitin sulfate proteoglycan (CSPG) core proteins in astrocytes treated with TGF-β. GM-CSF also inhibited the TGF-β-induced Rho-ROCK pathway, which is important in CSPG expression. Finally, the inhibitory effect of GM-CSF was blocked by a JAK inhibitor. These results may provide the basis for GM-CSF’s effects in glial scar inhibition and ultimately for its therapeutic effect on neural cell injuries. [BMB Reports 2014; 47(12): 679-684]     

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 promotes axonal re-growth and behavior recovery in spinal cord injury

In spinal cord injury, the injury could trigger some inhibitory signal cascades to promote chondroitin sulfate proteoglycans (CSPGs), the structures of scar tissues, formation. CSPGs could limit axonal regeneration mainly through the glycosaminoglycan (GAG) chain in the lesion site were suggested. We hypothesized that the digestion of CSPGs by chondroitinase ABC (ChABC) might decrease the inhibitory effects of limiting axonal re-growth after spinal cord injury. We compared the digesting products of CSPGs such as 2B6 by ChABC with the untreated control group and found no immunostaining of 2B6 in control group. The smaller size scars of ChABC-treatment were observed via CS-56, a type of CSPGs, 8 weeks after transection by immunohistochemistry. The inhibitory effects of CSPGs withdraw GAGs following ChABC-treatment would reduce, and immunopositive GAP-43 newly outgrown fibers were identified. In the animal trials, ChABC-treatment could improve motor function through BBB locomotor's test and reduce limiting ability of scar tissues to promote axonal regeneration via changing the structure of CSPGs by immunohistochemistry with GAP-43.     

Immature astrocytes promote CNS axonal regeneration when combined with chondroitinase ABC

Regeneration of injured adult CNS axons is inhibited by formation of a glial scar. Immature astrocytes are able to support robust neurite outgrowth and reduce scarring, therefore, we tested whether these cells would have this effect if transplanted into brain injuries. Utilizing an in vitro spot gradient model that recreates the strongly inhibitory proteoglycan environment of the glial scar we found that, alone, immature, but not mature, astrocytes had a limited ability to form bridges across the most inhibitory outer rim. In turn, the astrocyte bridges could promote adult sensory axon re‐growth across the gradient. The use of selective enzyme inhibitors revealed that MMP‐2 enables immature astrocytes to cross the proteoglycan rim. The bridge‐building process and axon regeneration across the immature glial bridges were greatly enhanced by chondroitinase ABC pretreatment of the spots. We used microlesions in the cingulum of the adult rat brains to test the ability of matrix modification and immature astrocytes to form a bridge for axon regeneration in vivo. Injured axons were visualized via p75 immunolabeling and the extent to which these axons regenerated was quantified. Immature astrocytes coinjected with chondroitinase ABC‐induced axonal regeneration beyond the distal edge of the lesion. However, when used alone, neither treatment was capable of promoting axonal regeneration. Our findings indicate that when faced with a minimal lesion, neurons of the basal forebrain can regenerate in the presence of a proper bridge across the lesion and when levels of chondroitin sulfate proteoglycans (CSPGs) in the glial scar are reduced. © 2010 Wiley Periodicals, Inc.Develop Neurobiol 70: 826–841, 2010     

MicroRNAs in the axon locally mediate the effects of chondroitin sulfate proteoglycans and cGMP on axonal growth

Axonal miRNAs locally regulate axonal growth by modulating local protein composition. Whether localized miRNAs in the axon mediate the inhibitory effect of Chondroitin sulfate proteoglycans (CSPGs) on the axon remains unknown. We showed that in cultured cortical neurons, axonal application of CSPGs inhibited axonal growth and altered axonal miRNA profiles, whereas elevation of axonal cyclic guanosine monophosphate (cGMP) levels by axonal application of sildenafil reversed the effect of CSPGs on inhibition of axonal growth and on miRNA profiles. Specifically, CSPGs elevated and reduced axonal levels of miR‐29c and integrin β1 (ITGB1) proteins, respectively, while elevation of cGMP levels overcame these CSPG effects. Gain‐of‐ and loss‐of‐function experiments demonstrated that miR‐29c in the distal axon mediates axonal growth downstream of CSPGs and cGMP by regulating axonal protein levels of ITGB1, FAK, and RhoA. Together, our data demonstrate that axonal miRNAs play an important role in mediating the inhibitory action of CSPGs on axonal growth and that miR‐29c at least partially mediates this process. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1402–1419, 2015     

Sustained delivery of activated Rho GTPases and BDNF promotes axon growth in CSPG-rich regions following spinal cord injury

Background

Spinal cord injury (SCI) often results in permanent functional loss. This physical trauma leads to secondary events, such as the deposition of inhibitory chondroitin sulfate proteoglycan (CSPG) within astroglial scar tissue at the lesion.

Methodology/Principal Findings

We examined whether local delivery of constitutively active (CA) Rho GTPases, Cdc42 and Rac1 to the lesion site alleviated CSPG-mediated inhibition of regenerating axons. A dorsal over-hemisection lesion was created in the rat spinal cord and the resulting cavity was conformally filled with an in situ gelling hydrogel combined with lipid microtubes that slowly released constitutively active (CA) Cdc42, Rac1, or Brain-derived neurotrophic factor (BDNF). Treatment with BDNF, CA-Cdc42, or CA-Rac1 reduced the number of GFAP-positive astrocytes, as well as CSPG deposition, at the interface of the implanted hydrogel and host tissue. Neurofilament 160kDa positively stained axons traversed the glial scar extensively, entering the hydrogel-filled cavity in the treatments with BDNF and CA-Rho GTPases. The treated animals had a higher percentage of axons from the corticospinal tract that traversed the CSPG-rich regions located proximal to the lesion site.

Conclusion

Local delivery of CA-Cdc42, CA-Rac1, and BDNF may have a significant therapeutic role in overcoming CSPG-mediated regenerative failure after SCI.     

Lentiviral vector delivery of short hairpin RNA to NG2 and neurotrophin-3 promotes locomotor recovery in injured rat spinal cord

Background aimsIn this study we investigated the effect of neurotrophin-3 (NT-3) and knockdown of NG2, one of the main inhibitory chondroitin sulfate proteoglycans (CSPG), in the glial scar following spinal cord injury (SCI).MethodsShort hairpin (sh) RNA were designed to target NG2 and were cloned into a lentiviral vector (LV). A LV was also constructed containing NT-3. LV expressing NT-3, shRNA to NG2 or combinations of both vectors were injected directly into contused rat spinal cords 1 week post-injury. Six weeks post-injection of LV, spinal cords were examined by histology for changes in scar size and by immunohistochemistry for changes in expression of CSPG, NT-3, astrocytes, neurons and microglia/macrophages. Motor function was assessed using the Basso, Beattie and Bresnahan (BBB) locomotor scale.ResultsAnimals that received the combination treatment of LV shNG2 and LV NT-3 showed reduced scar size. These animals also showed an increase in levels of neurons and NG2, a decrease in levels of astrocytes and a significant functional recovery as assessed using the BBB locomotor scale at 2 weeks post-treatment.ConclusionsThe improvement in locomotor recovery and decrease in scar size shows the potential of this gene therapy approach as a therapeutic treatment for SCI.     

Transplantation of D15A-Expressing Glial-Restricted-Precursor-Derived Astrocytes Improves Anatomical and Locomotor Recovery after Spinal Cord Injury

The transplantation of neural stem/progenitor cells is a promising therapeutic strategy for spinal cord injury (SCI). In this study, we tested whether combination of neurotrophic factors and transplantation of glial-restricted precursor (GRPs)-derived astrocytes (GDAs) could decrease the injury and promote functional recovery after SCI. We developed a protocol to quickly produce a sufficiently large, homogenous population of young astrocytes from GRPs, the earliest arising progenitor cell population restricted to the generation of glia. GDAs expressed the axonal regeneration promoting substrates, laminin and fibronectin, but not the inhibitory chondroitin sulfate proteoglycans (CSPGs). Importantly, GDAs or its conditioned medium promoted the neurite outgrowth of dorsal root ganglion neurons in vitro. GDAs were infected with retroviruses expressing EGFP or multi-neurotrophin D15A and transplanted into the contused adult thoracic spinal cord at 8 days post-injury. Eight weeks after transplantation, the grafted GDAs survived and integrated into the injured spinal cord. Grafted GDAs expressed GFAP, suggesting they remained astrocyte lineage in the injured spinal cord. But it did not express CSPG. Robust axonal regeneration along the grafted GDAs was observed. Furthermore, transplantation of D15A-GDAs significantly increased the spared white matter and decreased the injury size compared to other control groups. More importantly, transplantation of D15A-GDAs significantly improved the locomotion function recovery shown by BBB locomotion scores and Tredscan footprint analyses. However, this combinatorial strategy did not enhance the aberrant synaptic connectivity of pain afferents, nor did it exacerbate posttraumatic neuropathic pain. These results demonstrate that transplantation of D15A-expressing GDAs promotes anatomical and locomotion recovery after SCI, suggesting it may be an effective therapeutic approach for SCI.     

Mammalian-produced chondroitinase AC mitigates axon inhibition by chondroitin sulfate proteoglycans

Chondroitin sulfate proteoglycans (CSPGs) are up-regulated following spinal cord injury and are partly responsible for failed regeneration. Experimental paradigms in vivo that degrade chondroitin sulfate glycosaminoglycan chains with the bacterial enzyme, chondroitinase, greatly enhance the ability of axons to regenerate through the glial scar. Unfortunately, enthusiasm for this treatment paradigm is diminished by the lack of a minimally invasive and sustained delivery method. To address these deficits, we have engineered a Tet-On adenoviral vector encoding chondroitinase AC and have characterized its enzymatic function in vitro. U373 human astrocytoma cells were transduced with adenovirus and subsequently induced with doxycycline to secrete enzymatically active chondroitinase as detected by western blot and kinetic analyses. Enzymatic activity demonstrated biological relevance in studies where neurite outgrowth into and across CSPG-adsorbed regions pre-treated with conditioned media from chondroitinase secreting astrocytes was significantly increased compared with untreated controls (p < 0.0001). We also measured important parameters of enzyme activity including: pH, temperature, and enzyme stability that are fundamental to harnessing the true therapeutic potential of this approach. The use of resident cells for continuous secretion of CSPG-degrading enzymes at the site of the glial scar promises to be of greater clinical relevance than contemporary methods.     

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.     

NG2 and phosphacan are present in the astroglial scar after human traumatic spinal cord injury

Background

A major class of axon growth-repulsive molecules associated with CNS scar tissue is the family of chondroitin sulphate proteoglycans (CSPGs). Experimental spinal cord injury (SCI) has demonstrated rapid re-expression of CSPGs at and around the lesion site. The pharmacological digestion of CSPGs in such lesion models results in substantially enhanced axonal regeneration and a significant functional recovery. The potential therapeutic relevance of interfering with CSPG expression or function following experimental injuries seems clear, however, the spatio-temporal pattern of expression of individual members of the CSPG family following human spinal cord injury is only poorly defined. In the present correlative investigation, the expression pattern of CSPG family members NG2, neurocan, versican and phosphacan was studied in the human spinal cord.

Methods

An immunohistochemical investigation in post mortem samples of control and lesioned human spinal cords was performed. All patients with traumatic SCI had been clinically diagnosed as having "complete" injuries and presented lesions of the maceration type.

Results

In sections from control spinal cord, NG2 immunoreactivity was restricted to stellate-shaped cells corresponding to oligodendrocyte precursor cells. The distribution patterns of phosphacan, neurocan and versican in control human spinal cord parenchyma were similar, with a fine reticular pattern being observed in white matter (but also located in gray matter for phosphacan). Neurocan staining was also associated with blood vessel walls. Furthermore, phosphacan, neurocan and versican were present in the myelin sheaths of ventral and dorsal nerve roots axons. After human SCI, NG2 and phosphacan were both detected in the evolving astroglial scar. Neurocan and versican were detected exclusively in the lesion epicentre, being associated with infiltrating Schwann cells in the myelin sheaths of invading peripheral nerve fibres from lesioned dorsal roots.

Conclusion

NG2 and phosphacan were both present in the evolving astroglial scar and, therefore, might play an important role in the blockade of successful CNS regeneration. Neurocan and versican, however, were located at the lesion epicentre, associated with Schwann cell myelin on regenerating peripheral nerve fibres, a distribution that was unlikely to contribute to failed CNS axon regeneration. The present data points to the importance of such correlative investigations for demonstrating the clinical relevance of experimental data.     

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.