Chondroitin Sulfate Proteoglycan Core Protein Research Articles

Chondroitinase ABC Administration Facilitates Serotonergic Innervation of Motoneurons in Rats With Complete Spinal Cord Transection.

Chondroitinase ABC (ChABC) is an enzyme that degrades glycosaminoglycan side-chains of chondroitin sulfate (CS-GAG) from the chondroitin sulfate proteoglycan (CSPG) core protein. Previous studies demonstrated that the administration of ChABC after spinal cord injury promotes nerve regeneration by removing CS-GAGs from the lesion site and promotes the plasticity of spinal neurons by removing CS-GAGs from the perineuronal nets (PNNs). These effects of ChABC might enhance the regeneration and sprouting of descending axons, leading to the recovery of motor function. Anatomical evidence, indicating that the regenerated axons innervate spinal motoneurons caudal to the lesion site, however, has been lacking. In the present study, we investigated whether descending axons pass through the lesion site and innervate the lumbar motoneurons after ChABC administration in rats with complete spinal cord transection (CST) at the thoracic level. At 3 weeks after CST, 5-hydroxytryptamine (5-HT) fibers were observed to enter the lesion in ChABC-treated rats, but not saline-treated rats. In addition, 92% of motoneurons in the ventral horn of the fifth lumbar segment (L5) in saline-treated rats, and 38% of those in ChABC-treated rats were surrounded by chondroitin sulfate-A (CS-A) positive structures. At 8 weeks after CST, many 5-HT fibers were observed in the ventral horn of the L5, where they terminated in the motoneurons in ChABC-treated rats, but not in saline-treated rats. In total, 54% of motoneurons in the L5 ventral horn in saline-treated rats and 39% of those in ChABC-treated rats were surrounded by CS-A-positive structures. ChABC-treated rats had a Basso, Beattie, and Bresnahan (BBB) motor score of 3.8 at 2 weeks, 7.1 at 3 weeks, and 10.3 at 8 weeks after CST. These observations suggest that ChABC administration to the lesion site immediately after CST may promote the regeneration of descending 5-HT axons through the lesion site and their termination on motoneurons at the level of caudal to the lesion site. ChABC administration might facilitate reinnervation by degrading CS-GAGs around motoneurons. Motor function of the lower limbs was significantly improved in ChABC-treated rats even before the 5-HT axons terminated on the motoneurons, suggesting that other mechanisms may also contribute to the motor function recovery.

Regulation of perineuronal net components in the synaptic bouton vicinity on lumbar α-motoneurons in the rat after spinalization and locomotor training: New insights from spatio-temporal changes in gene, protein expression and WFA labeling

Chondroitin sulfate proteoglycans (CSPGs) consist of core proteins and glycosaminoglycan side chains. Tenascins, and hyaluronan and proteoglycan link protein 1 (HAPLN), link CSPGs with a hyaluronan backbone to constitute perineuronal nets (PNNs), which ensheath preferentially highly active neurons to maintain architecture and stabilize synapses, but restrict repair plasticity. Spinal cord injury increases CSPG core protein levels in the lesion proximity, limiting permissiveness of the extracellular milieu for fiber regrowth, however regulation of PNNs structure in the vicinity of distant α-motoneurons (MNs) in the course of degeneration and reorganization of their inputs requires research. Here, we examined early and late changes in CSPGs, HAPLN1, tenascin-R, and glial activation along the spinal cord in male rats with complete spinal cord transection (Th10), and their impact on PNNs ensheathing lumbar MNs innervating ankle extensor and flexor muscles, which are in different loading states in paraplegic rats. We show that (1) distance from the lesion site and time after injury (2–5 weeks) differentiate degree of changes in transcription rates (measured with RT-qPCR) of PNNs proteins with increased CSPGs and decreased HAPLN1 transcripts, suggesting long-term PNN destabilization in majority of spinal segments, (2) in lumbar segments PNN composition is not MN-class (extensor vs flexor) specific, both showing early decrease and late upregulation of Wisteria floribunda agglutinin (WFA) labeling in vicinity of synaptic boutons on MNs, (3) long-term locomotor training tends to reduce WFA(+) PNNs, but not their protein components (immunofluorescence measurements) around MNs. Our results suggest that training-induced regulation may target glycan structures of CSPGs.

Molecular Interactions Stabilizing the Promatrix Metalloprotease-9·Serglycin Heteromer

Previous studies have shown that THP-1 cells produced an SDS-stable and reduction-sensitive complex between proMMP-9 and a chondroitin sulfate proteoglycan (CSPG) core protein. The complex could be reconstituted in vitro using purified serglycin (SG) and proMMP-9 and contained no inter-disulfide bridges. It was suggested that the complex involved both the FnII module and HPX domain of proMMP-9. The aims of the present study were to resolve the interacting regions of the molecules that form the complex and the types of interactions involved. In order to study this, we expressed and purified full-length and deletion variants of proMMP-9, purified CSPG and SG, and performed in vitro reconstitution assays, peptide arrays, protein modelling, docking, and molecular dynamics (MD) simulations. ProMMP-9 variants lacking both the FnII module and the HPX domain did not form the proMMP-9∙CSPG/SG complex. Deletion variants containing at least the FnII module or the HPX domain formed the proMMP-9∙CSPG/SG complex, as did the SG core protein without CS chains. The interacting parts covered large surface areas of both molecules and implicated dynamic and complementary ionic, hydrophobic, and hydrogen bond interactions. Hence, no short single interacting linear motifs in the two macromolecules could explain the strong SDS-stable and reduction-sensitive binding.

Chondroitin sulfate content and decorin expression in glioblastoma are associated with proliferative activity of glioma cells and disease prognosis.

Chondroitin sulfate proteoglycans (CSPGs) are important components of brain extracellular matrix (ECM), although their contribution in gliomagenesis remains underinvestigated. Here, both chondroitin sulfate (CS) content/distribution and expression of a number of CSPG core proteins were studied in glioblastoma multiforme (GBM) tumours with different prognosis (n = 40) using immunohistochemistry and RT-PCR analysis. Survival rates for clinically different patient groups were compared using the Kaplan-Meier analysis and univariate Cox model. CS content was increased in 60-65% of studied GBM tumours and distributed heterogeneously, mainly at perinecrotic and perivascular zones rather than tumour cells with specific morphology. CS accumulation, especially in the tumour extracellular matrix, was positively associated with the proliferative activity of GBM cells according to theKi67 index (p < 0.01) but revealed no significant association with age or sex of the patients, tumour localisation, relapse or disease outcome. The increase in CS content in GBM tumours was accompanied by upregulation of decorin (1.5-fold), biglycan (3-fold) and serglycin (2-fold) expression (p < 0.05), while only decorin expression level was negatively associated with the overall survival rate of the GBM patients (p < 0.05). These results demonstrate a contribution of CS to high intratumoural heterogeneity of GBM and suggest CS content and decorin expression for further investigation as potential microenvironmental glycomarkers/targets for GBM diagnostics and treatment.

Spatiotemporal distribution of chondroitin sulfate proteoglycans after optic nerve injury in rodents

The accumulation of chondroitin sulfate proteoglycans (CSPGs) in the glial scar following acute damage to the central nervous system (CNS) limits the regeneration of injured axons. Given the rich diversity of CSPG core proteins and patterns of GAG sulfation, identifying the composition of these CSPGs is essential for understanding their roles in injury and repair. Differential expression of core proteins and sulfation patterns have been characterized in the brain and spinal cord of mice and rats, but a comprehensive study of these changes following optic nerve injury has not yet been performed. Here, we show that the composition of CSPGs in the optic nerve and retina following optic nerve crush (ONC) in mice and rats exhibits an increase in aggrecan, brevican, phosphacan, neurocan and versican, similar to changes following spinal cord injury. We also observe an increase in inhibitory 4-sulfated (4S) GAG chains, which suggests that the persistence of CSPGs in the glial scar opposes the growth of CNS axons, thereby contributing to the failure of regeneration and recovery of function.

Expanding the chondroitin glycoproteome of Caenorhabditis elegans

Chondroitin sulfate proteoglycans (CSPGs) are important structural components of connective tissues in essentially all metazoan organisms. In vertebrates, CSPGs are involved also in more specialized processes such as neurogenesis and growth factor signaling. In invertebrates, however, knowledge of CSPGs core proteins and proteoglycan-related functions is relatively limited, even for Caenorhabditis elegans. This nematode produces large amounts of non-sulfated chondroitin in addition to low-sulfated chondroitin sulfate chains. So far, only nine core proteins (CPGs) have been identified, some of which have been shown to be involved in extracellular matrix formation. We recently introduced a protocol to characterize proteoglycan core proteins by identifying CS-glycopeptides with a combination of biochemical enrichment, enzymatic digestion, and nano-scale liquid chromatography MS/MS analysis. Here, we have used this protocol to map the chondroitin glycoproteome in C. elegans, resulting in the identification of 15 novel CPG proteins in addition to the nine previously established. Three of the newly identified CPGs displayed homology to vertebrate proteins. Bioinformatics analysis of the primary protein sequences revealed that the CPG proteins altogether contained 19 unique functional domains, including Kunitz and endostatin domains, suggesting direct involvement in protease inhibition and axonal migration, respectively. The analysis of the core protein domain organization revealed that all chondroitin attachment sites are located in unstructured regions. Our results suggest that CPGs display a much greater functional and structural heterogeneity than previously appreciated and indicate that specialized proteoglycan-mediated functions evolved early in metazoan evolution.

Abnormalities in perineuronal nets and behavior in mice lacking CSGalNAcT1, a key enzyme in chondroitin sulfate synthesis

Chondroitin sulfate (CS) is an important glycosaminoglycan and is mainly found in the extracellular matrix as CS proteoglycans. In the brain, CS proteoglycans are highly concentrated in perineuronal nets (PNNs), which surround synapses and modulate their functions. To investigate the importance of CS, we produced and precisely examined mice that were deficient in the CS synthesizing enzyme, CSGalNAcT1 (T1KO). Biochemical analysis of T1KO revealed that loss of this enzyme reduced the amount of CS by approximately 50% in various brain regions. The amount of CS in PNNs was also diminished in T1KO compared to wild-type mice, although the amount of a major CS proteoglycan core protein, aggrecan, was not changed. In T1KO, we observed abnormalities in several behavioral tests, including the open-field test, acoustic startle response, and social preference. These results suggest that T1 is important for plasticity, probably due to regulation of CS-dependent PNNs, and that T1KO is a good model for investigation of PNNs.

Placental Sequestration of Plasmodium falciparum Malaria Parasites Is Mediated by the Interaction Between VAR2CSA and Chondroitin Sulfate A on Syndecan-1

During placental malaria, Plasmodium falciparum infected erythrocytes sequester in the placenta, causing health problems for both the mother and fetus. The specific adherence is mediated by the VAR2CSA protein, which binds to placental chondroitin sulfate (CS) on chondroitin sulfate proteoglycans (CSPGs) in the placental syncytium. However, the identity of the CSPG core protein and the cellular impact of the interaction have remain elusive. In this study we identified the specific CSPG core protein to which the CS is attached, and characterized its exact placental location. VAR2CSA pull-down experiments using placental extracts from whole placenta or syncytiotrophoblast microvillous cell membranes showed three distinct CSPGs available for VAR2CSA adherence. Further examination of these three CSPGs by immunofluorescence and proximity ligation assays showed that syndecan-1 is the main receptor for VAR2CSA mediated placental adherence. We further show that the commonly used placental choriocarcinoma cell line, BeWo, express a different set of proteoglycans than those present on placental syncytiotrophoblast and may not be the most biologically relevant model to study placental malaria. Syncytial fusion of the BeWo cells, triggered by forskolin treatment, caused an increased expression of placental CS-modified syndecan-1. In line with this, we show that rVAR2 binding to placental CS impairs syndecan-1-related Src signaling in forskolin treated BeWo cells, but not in untreated cells.

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 glycosaminoglycans for CNS homeostasis-implications for material design.

Chondroitin sulfate proteoglycans (CSPGs) are complex biomolecules that are known to facilitate patterning of axonal direction and cell migration during the early growth and development phase of the mammalian central nervous system (CNS). In adults, they continue to control neuronal plasticity as major constituents of the "peri-neuronal nets" (PNNs) that surround adult CNS neurons. CSPGs are also barrier-forming molecules that are selectively upregulated by invading reactive astroglia after injury to the CNS, and are responsible for the active repulsion of regenerating neurons post-injury. Recent evidence however suggests that the diverse sulfated glycosaminoglycan (GAG) side chains attached to CSPGs are key components that play paradoxical roles in influencing nerve regeneration post-injury to the CNS. Sulfated GAG repeats attached to the CSPG core protein help mediate cell migration, neuritogenesis, axonal pathfinding, and axonal repulsion by directly trapping and presenting a whole host of growth factors to cells locally, or by binding to specific membrane bound proteins on the cell surface to influence cellular function. In this review, we will present the current gamut of interventional strategies used to bridge CNS deficits, and discuss the potential advantages of using sulfated GAG based biomaterials to facilitate the repair and regeneration of the injured CNS.

OASIS regulates chondroitin 6‐O‐sulfotransferase 1 gene transcription in the injured adult mouse cerebral cortex

Old astrocyte specifically induced substance (OASIS), a basic leucine zipper transcription factor of the cAMP response element binding/Activating transcription factor family, is induced in reactive astrocytes in vivo and has important roles in quality control of protein synthesis at the endoplasmic reticulum. Reactive astrocytes produce a non-permissive environment for regenerating axons by up-regulating chondroitin sulfate proteoglycans (CSPGs). In this study, we focus on the potential role of OASIS in CSPG production in the adult mouse cerebral cortex. CS-C immunoreactivity, which represents chondroitin sulfate moieties, was significantly attenuated in the stab-injured cortices of OASIS knockout mice compared to those of wild-type mice. We next examined expression of the CSPG-synthesizing enzymes and core proteins of CSPGs in the stab-injured cortices of OASIS knockout and wild-type mice. The levels of chondroitin 6-O-sulfotransferase 1 (C6ST1, one of the major enzymes involved in sulfation of CSPGs) mRNA and protein increased after cortical stab injury of wild-type, but not of OASIS knockout, mice. A C-terminal deletion mutant OASIS over-expressed in rat C6 glioma cells increased C6ST1 transcription by interacting with the first intron region. Neurite outgrowth of cultured hippocampal neurons was inhibited on culture dishes coated with membrane fractions of epidermal growth factor-treated astrocytes derived from wild type but not from OASIS knockout mice. These results suggest that OASIS regulates the transcription of C6ST1 and thereby promotes CSPG sulfation in astrocytes. Through these mechanisms, OASIS may modulate axonal regeneration in the injured cerebral cortex. OASIS, an ER stress-responsive CREB/ATF family member, is up-regulated in the reactive astrocytes of the injured brain. We found that the up-regulated OASIS is involved in the transcriptional regulation of C6ST1 gene, which promotes chondroitin sulfate proteoglycan (CSPG) sulfation. We conclude that OASIS functions as an anti-regenerative transcription factor by establishing a non-permissive microenvironment to regenerating axons.

In vitro reconstitution of complexes between pro‐matrix metalloproteinase‐9 and the proteoglycans serglycin and versican

Previously, we have shown that a proportion of the matrix metalloproteinase-9 (MMP-9) synthesized by the macrophage cell line THP-1 binds to a chondroitin sulfate proteoglycan (CSPG) core protein to form a reduction-sensitive heteromer. It was also shown that the hemopexin-like (PEX) domain and the fibronectin-like (FnII) module in the enzyme are involved in heteromer formation. In this paper, we show that reduction-sensitive and SDS-stable heteromers may be reconstituted in vitro by mixing proMMP-9 with either serglycin, versican or CSPGs isolated from various monocytic cell lines. In addition, a strong but SDS-soluble proMMP-9·CSPG heteromer was formed. The two macromolecules in the SDS-stable reduction-sensitive heteromers were not linked together by disulfide bonds. As for the heteromer isolated from THP-1 cells, in vitro reconstituted SDS-stable and SDS-soluble heteromers showed weaker binding to gelatin than the proMMP-9 monomer. Furthermore, gelatin inhibited in vitro reconstitution of the heteromers, showing that the FnII module is involved in the complex formation. Tissue inhibitor of metalloproteinase (TIMP)-1 was not be detected in the proMMP-9·CSPG complexes. However, the presence of TIMP-1 inhibited formation of the SDS-soluble heteromer, but not the SDS-stable reduction-sensitive heteromer. This indicates that different regions in the PEX domain are involved formation of these heteromers.

Overcoming neurite‐inhibitory chondroitin sulfate proteoglycans in the astrocyte matrix

Acute trauma to the central nervous system (CNS) can result in permanent damage and loss of function related to the poor regeneration of injured axons. Injured axons encounter several barriers to regeneration, such as the glial scar at the injury site. The glial scar contains extracellular matrix (ECM) macromolecules deposited by reactive astrocytes in response to injury. The scar ECM is rich in chondroitin sulfate proteoglycans (CSPGs), macromolecules that inhibit axonal growth. CSPGs consist of a core protein with attachment sites for glycosaminoglycan (GAG) chains. An extensive literature demonstrates that enzymatic removal of the GAG chains by chondroitinase ABC permits some axonal regrowth; however, the remaining intact core proteins also possess inhibitory domains. Because metalloproteinases can degrade core proteins of CSPGs, we have evaluated five matrix metalloproteinases (MMPs) and a related protease-a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4)-for their capacity to overcome CSPG inhibition of neuritic growth in culture. The metalloproteinases were selected for their known expression after CNS injuries. Of the MMPs, MMP-3, -7 and -8 reduced or abolished inhibition of neurite outgrowth on a purified CSPG substrate and on an astrocyte-derived ECM. ADAMTS-4 also attenuated CSPG inhibition of neurites and had the additional benefits of neither degrading laminin nor causing neurotoxicity. The efficacy of ADAMTS-4 matched that of blocking the EGFR signaling previously reported to mediate CSPG inhibition. These findings highlight ADAMTS-4 as a superior protease for overcoming CSPG inhibition of axonal regeneration in the CNS.

Conditional Sox9 ablation reduces chondroitin sulfate proteoglycan levels and improves motor function following spinal cord injury

Chondroitin sulfate proteoglycans (CSPGs) found in perineuronal nets and in the glial scar after spinal cord injury have been shown to inhibit axonal growth and plasticity. Since we have previously identified SOX9 as a transcription factor that upregulates the expression of a battery of genes associated with glial scar formation in primary astrocyte cultures, we predicted that conditional Sox9 ablation would result in reduced CSPG expression after spinal cord injury and that this would lead to increased neuroplasticity and improved locomotor recovery. Control and Sox9 conditional knock-out mice were subject to a 70 kdyne contusion spinal cord injury at thoracic level 9. One week after injury, Sox9 conditional knock-out mice expressed reduced levels of CSPG biosynthetic enzymes (Xt-1 and C4st), CSPG core proteins (brevican, neurocan, and aggrecan), collagens 2a1 and 4a1, and Gfap, a marker of astrocyte activation, in the injured spinal cord compared with controls. These changes in gene expression were accompanied by improved hind limb function and locomotor recovery as evaluated by the Basso Mouse Scale (BMS) and rodent activity boxes. Histological assessments confirmed reduced CSPG deposition and collagenous scarring at the lesion of Sox9 conditional knock-out mice, and demonstrated increased neurofilament-positive fibers in the lesion penumbra and increased serotonin immunoreactivity caudal to the site of injury. These results suggest that SOX9 inhibition is a potential strategy for the treatment of SCI.

Alterations in sulfated chondroitin glycosaminoglycans following controlled cortical impact injury in mice

Chondroitin sulfate proteoglycans (CSPGs) play a pivotal role in many neuronal growth mechanisms including axon guidance and the modulation of repair processes following injury to the spinal cord or brain. Many actions of CSPGs in the central nervous system (CNS) are governed by the specific sulfation pattern on the glycosaminoglycan (GAG) chains attached to CSPG core proteins. To elucidate the role of CSPGs and sulfated GAG chains following traumatic brain injury (TBI), controlled cortical impact injury of mild to moderate severity was performed over the left sensory motor cortex in mice. Using immunoblotting and immunostaining, we found that TBI resulted in an increase in the CSPGs neurocan and NG2 expression in a tight band surrounding the injury core, which overlapped with the presence of 4-sulfated CS GAGs but not with 6-sulfated GAGs. This increase was observed as early as 7 days post injury (dpi), and persisted for up to 28 dpi. Labeling with markers against microglia/macrophages, NG2+ cells, fibroblasts, and astrocytes showed that these cells were all localized in the area, suggesting multiple origins of chondroitin-4-sulfate increase. TBI also caused a decrease in the expression of aggrecan and phosphacan in the pericontusional cortex with a concomitant reduction in the number of perineuronal nets. In summary, we describe a dual response in CSPGs whereby they may be actively involved in complex repair processes following TBI.

Multiple enhancers associated with ACAN suggest highly redundant transcriptional regulation in cartilage

The chondroitin sulfate proteoglycan core protein aggrecan is the major protein constituent of cartilage aside from collagen, and is largely responsible for its distinctive mechanical properties. Aggrecan is required both for proper cartilage formation in development and maintenance of mature cartilage. Prominent ACAN transcription is a conserved feature of vertebrate cartilage, although little is known about its specific transcriptional regulation. We examined the genomic interval containing human ACAN for transcriptional enhancers directing expression to cartilage, using a functional assay in transgenic zebrafish. We tested 24 conserved non‐coding sequences, representing ~6% of the total sequence in the interval, and identified eleven independently capable of regulating reporter gene expression in cartilage. These enhancers were widely spaced, from >100kb upstream of the gene to within the first intron. While the majority displayed broad cartilage expression in zebrafish larvae, several were restricted to a subset of cartilage cells in the craniofacial skeleton. In older fish, the enhancers displayed differential activity; some maintained expression, either in all cartilage or preferentially in articular cartilage at the joints, while others were not active. This remarkable degree of overlapping regulatory control has been highly conserved; we identified clear orthologues of six enhancers at the chicken ACAN locus, arranged in the same order relative to the gene. These were also functional in directing expression to cartilage in transgenic zebrafish. Several enhancers contain potential binding sites for Sox9, consistent with its described role as an upstream regulator of ACAN expression. However, others lacked Sox9 consensus binding sites, implicating additional pathways and transcription factors as regulators of ACAN expression in cartilage, either in development or adult tissue. Our identification of these enhancer sequences is the necessary first step in detailed examination of the upstream regulators of ACAN expression.

Gliosis-specific transcription factor OASIS coincides with proteoglycan core protein genes in the glial scar and inhibits neurite outgrowth

OASIS gene, a member of the CREB/ATF transcription factor family, is upregulated in gliosis after CNS injury. However it remains to be determined how OASIS is implicated in gliotic reaction. In a glial scar, chondroitin sulfate proteoglycans (CSPGs) are also upregulated, which engenders the inhibition of axonal regeneration. We investigated the functional role of OASIS in gliosis in relation to CSPG core proteins that render lesions non-permissive for regenerating axons. We first examined the gene expression localization of OASIS using several markers in a cryo-injured mouse brain and compared the expression pattern of CSPG core protein genes with that of OASIS in a glial scar by double-labeling in situ hybridization. Our findings suggest that OASIS is induced in proximal reactive astrocytes that exhibit upregulated expression for CSPGs, including NG2 proteoglycan, versican, brevican, neurocan, and phosphacan core. Furthermore, the membrane fraction derived from OASIS-transfected C6 cells inhibits neurite outgrowth of NG108-15 cells, whereas its neurite outgrowth inhibitory effect is abrogated after chondroitinase ABC treatment. OASIS is likely to be involved in the regulatory mechanism of non-permissive environments for axonal outgrowth.

Ultraviolet Irradiation Induces the Accumulation of Chondroitin Sulfate, but Not Other Glycosaminoglycans, in Human Skin

Ultraviolet (UV) light alters cutaneous structure and function. Prior work has shown loss of dermal hyaluronan after UV-irradiation of human skin, yet UV exposure increases total glycosaminoglycan (GAG) content in mouse models. To more fully describe UV-induced alterations to cutaneous GAG content, we subjected human volunteers to intermediate-term (5 doses/week for 4 weeks) or single-dose UV exposure. Total dermal uronyl-containing GAGs increased substantially with each of these regimens. We found that UV exposure substantially increased dermal content of chondroitin sulfate (CS), but not hyaluronan, heparan sulfate, or dermatan sulfate. UV induced the accumulation of both the 4-sulfated (C4S) and 6-sulfated (C6S) isoforms of CS, but in distinct distributions. Next, we examined several CS proteoglycan core proteins and found a significant accumulation of dermal and endothelial serglycin, but not of decorin or versican, after UV exposure. To examine regulation in vitro, we found that UVB in combination with IL-1α, a cytokine upregulated by UV radiation, induced serglycin mRNA in cultured dermal fibroblasts, but did not induce the chondroitin sulfate synthases. Overall, our data indicate that intermediate-term and single-dose UVB exposure induces specific GAGs and proteoglycan core proteins in human skin in vivo. These molecules have important biologic functions and contribute to the cutaneous response to UV.

Accumulation of chondroitin sulfate proteoglycans in the microenvironment of spinal motor neurons in amyotrophic lateral sclerosis transgenic rats

Chondroitin sulfate proteoglycans (CSPGs) are the major components of extracellular matrix in the central nervous system. In the spinal cord under various types of injury, reactive gliosis emerges in the lesion accompanied by CSPG up-regulation. Several types of CSPG core proteins and their side chains have been shown to inhibit axonal regeneration in vitro and in vivo. In the present study, we examined spatiotemporal expression of CSPGs in the spinal cord of transgenic (Tg) rats with His46Arg mutation in the Cu/Zn superoxide dismutase gene, a model of amyotrophic lateral sclerosis (ALS). Immunofluorescence disclosed a significant up-regulation of neurocan, versican, and phosphacan in the ventral spinal cord of Tg rats compared with age-matched controls. Notably, Tg rats showed progressive and prominent accumulation of neurocan even at the presymptomatic stage. Immunoblotting confirmed the distinct increase in the levels of both the full-length neurocan and their fragment isoforms. On the other hand, the up-regulation of versican and phosphacan peaked at the early symptomatic stage, followed by diminishment at the late symptomatic stage. In addition, double immunofluorescence revealed a colocalization between reactive astrocytes and immunoreactivities for neurocan and phosphacan, especially around residual large ventral horn neurons. Thus, reactive astrocytes are suggested to be participants in the CSPG accumulation. Although the possible neuroprotective involvement of CSPG remains to be investigated, the present results suggest that both the reactive astrocytes and the differential accumulation of CSPGs may create a nonpermissive microenvironment for neural regeneration in neurodegenerative diseases such as ALS.

TPA‐mediated generation of plasmin is catalyzed by the proteoglycan NG2

Paralysis resulting from spinal cord injury is devastating and persistent. One major reason for the inability of the body to heal this type of injury ensues from the local increase of glial cells leading to the formation of a glial scar, and the upregulation of chondroitin sulfate proteoglycans (CSPGs) at the site of injury through which axons are unable to regenerate. Experimental approaches to overcome this problem have accordingly focused on reducing the inhibitory properties of CSPGs, for example by using chondroitinase to remove the sugar chains and reduce the CSPGs to their core protein constituents, although this step alone does not provide dramatic benefits as a monotherapy. Using in vitro and in vivo approaches, we describe here a potentially synergistic therapeutic opportunity based on tissue plasminogen activator (tPA), an extracellular protease that converts plasminogen (plg) into the active protease plasmin. We show that tPA and plg both bind to the CSPG protein NG2, which functions as a scaffold to accelerate the tPA-driven conversion of plg to plasmin. The binding occurs via the tPA and plg kringle domains to domain 2 of the NG2 CSPG core protein, and is enhanced in some settings after chondroitinase-mediated removal of the NG2 proteoglycan side chains. Once generated, plasmin then degrades NG2, both in an in vitro setting using recombinant protein, and in vivo models of spinal cord injury. Our finding that the tPA and plg binding is in some instances more efficient after exposure of the NG2 proteoglycan to chondroitinase treatment suggests that a combined therapeutic approach employing both chondroitinase and the tPA/plasmin proteolytic system could be of significant benefit in promoting axonal regeneration through glial scars after spinal cord injury.