Chondroitin Sulfate Proteoglycans Neurocan Research Articles

The Chondroitin Sulfate Proteoglycans Neurocan and Phosphacan Are Expressed by Reactive Astrocytes in the Chronic CNS Glial Scar

Chondroitin sulfate proteoglycans (CS-PGs) expressed by reactive astrocytes may contribute to the axon growth-inhibitory environment of the injured CNS. The specific potentially inhibitory CS-PGs present in areas of reactive gliosis, however, have yet to be thoroughly examined. In this study, we used immunohistochemistry, combined immunohistochemistry-in situ hybridization, immunoblot analysis, and reverse transcription-PCR to examine the expression of specific CS-PGs by reactive astrocytes in an in vivo model of reactive gliosis: that is, the glial scar, after cortical injury. Neurocan and phosphacan can be localized to reactive astrocytes 30 d after CNS injury, whereas brevican and versican are not expressed in the chronic glial scar. Neurocan is also expressed by astrocytes in primary cell culture. Relative to the amount present in cultured astrocytes or uninjured cortex, neurocan expression increases significantly in the glial scar resulting from cortical injury, including the re-expression of the neonatal isoform of neurocan. In contrast, phosphacan protein levels are decreased in the glial scar compared with the uninjured brain. Because these CS-PGs are capable of inhibiting neurite outgrowth in vitro, our data suggest that phosphacan and neurocan in areas of reactive gliosis may contribute to axonal regenerative failure after CNS injury.

Entorhinal Cortex Lesion in Adult Rats Induces the Expression of the Neuronal Chondroitin Sulfate Proteoglycan Neurocan in Reactive Astrocytes

The chondroitin sulfate proteoglycan neurocan is a major component of brain extracellular matrix during development. Neurocan is primarily synthesized by neurons and has the ability to interact with cell adhesion molecules involved in the regulation of cell migration and axonal growth. Within the first weeks postnatally, neurocan expression is strongly downregulated. To test whether neurocan is reexpressed in areas of axonal growth (sprouting) after brain injury, the time course of neurocan expression was analyzed in the denervated fascia dentata of the rat after entorhinal cortex lesion (12 hr; 1, 2, 4, and 10 d; 2 and 4 weeks; and 6 months after lesion). In the denervated zone, immunohistochemistry revealed neurocan-positive astrocytes by 2 d after lesion and a diffuse labeling of the extracellular matrix at all later time points. Electron microscopy confirmed the deposition of neurocan in the extracellular matrix compartment. In situ hybridization demonstrated a strong upregulation of neurocan mRNA within the denervated outer molecular layer 1 and 4 d after lesion. The combination of in situ hybridization with immunohistochemistry for glial fibrillary acidic protein demonstrated that the neurocan mRNA-expressing cells are astrocytes. These data demonstrate that neurocan is reexpressed in the injured brain. In contrast to the situation during development, astrocytes, but not neurons, express neurocan and enrich the extracellular matrix with this molecule. Similar to the situation during development, neurocan is expressed in an area of active axon growth, and it is suggested that neurocan acts to maintain the boundaries of the denervated fascia dentata after entorhinal cortex lesion.

Transient compartmental expression of neurocan in the developing striatum of the rat.

The expression of the chondroitin sulfate proteoglycan neurocan was examined in the developing striatum of the rat and compared with the distribution of dopaminergic terminals. Neurocan immunoreactivity shows a homogeneous pattern in the embryonic striatum. In the postnatal striatum, neurocan was first expressed within the matrix but not the patch compartments, and subsequently within both. These results suggest that chondroitin sulfate proteoglycans are involved in formation of connections between the substantia nigra and striatum.

High Affinity Binding and Overlapping Localization of Neurocan and Phosphacan/Protein-tyrosine Phosphatase-ζ/β with Tenascin-R, Amphoterin, and the Heparin-binding Growth-associated Molecule

We have studied the interactions of the nervous tissue-specific chondroitin sulfate proteoglycans neurocan and phosphacan with the extracellular matrix protein tenascin-R and two heparin-binding proteins, amphoterin and the heparin-binding growth-associated molecule (HB-GAM), using a radioligand binding assay. Both proteoglycans show saturable, high affinity binding to tenascin-R with apparent dissociation constants in the 2-7 nM range. Binding is reversible, inhibited in the presence of unlabeled proteoglycan, and increased by approximately 60% following chondroitinase treatment of the proteoglycans, indicating that the interactions are mediated via the core (glyco)proteins rather than by the glycosaminoglycan chains, which may in fact partially shield the binding sites. In contrast to their interactions with tenascin-C, in which binding was decreased by approximately 75% in the absence of calcium, binding of phosphacan to tenascin-R was not affected by the absence of divalent cations in the binding buffer, although there was a small but significant decrease in the binding of neurocan. Neurocan and phosphacan are also high affinity ligands of amphoterin and HB-GAM (Kd = 0.3-8 nM), two heparin-binding proteins that are developmentally regulated in brain and functionally involved in neurite outgrowth. The chondroitin sulfate chains on neurocan and phosphacan account for at least 80% of their binding to amphoterin and HB-GAM. The presence of amphoterin also produces a 5-fold increase in phosphacan binding to the neural cell adhesion molecule contactin. Immunocytochemical studies showed an overlapping localization of the proteoglycans and their ligands in the embryonic and postnatal brain, retina, and spinal cord. These studies have therefore revealed differences in the interactions of neurocan and phosphacan with the two major members of the tenascin family of extracellular matrix proteins, and also suggest that chondroitin sulfate proteoglycans play an important role in the binding and/or presentation of differentiation factors in the developing central nervous system.

Isolation of a Tenascin-R Binding Protein from Mouse Brain Membranes: A PHOSPHACAN-RELATED CHONDROITIN SULFATE PROTEOGLYCAN

We have isolated a chondroitin sulfate proteoglycan from mouse brain by affinity chromatography with a fragment of the extracellular matrix glycoprotein tenascin-R (TN-R) that comprises the amino-terminal cysteine-rich stretch and the 4.5 epidermal growth factor-like repeats. The isolated chondroitin sulfate proteoglycan has a molecular mass of 500-600 kDa and carries the HNK-1 carbohydrate epitope. Treatment with chondroitinase ABC reveals a major band of approximately 400 kDa and two minor bands at 200 and 150 kDa. Immunoblot analysis relates the molecule to phosphacan but not to the chondroitin sulfate proteoglycans neurocan and versican. Binding of the phosphacan-related molecule to the epidermal growth factor-like repeats of TN-R is Ca2+-dependent. Co-localization of the molecule with TN-R in the retina and optic nerve by immunocytochemistry suggests a functional relationship between the two molecules in vivo. Inhibition of neurite outgrowth from hippocampal neurons by the phosphacan-related molecule in vitro is neutralized by TN-R when coated as a uniform substrate. Furthermore, the phosphacan-related molecule neutralizes growth cone repulsion induced by TN-R coated as a sharp substrate boundary with or without prior treatment with chondroitinase ABC. These observations indicate that TN-R can interact with a phosphacan-related molecule and thereby modulate its inhibitory influence on neuritogenesis.

TAG-1/Axonin-1 Is a High-affinity Ligand of Neurocan, Phosphacan/Protein-tyrosine Phosphatase-ζ/β, and N-CAM

Proteoglycans appear to play an important role in modulating cell-cell and cell-matrix interactions during nervous tissue histogenesis. The nervous tissue-specific chondroitin sulfate proteoglycans neurocan and phosphacan/protein-tyrosine phosphatase-zeta/beta were found to be high-affinity ligands of the neural cell adhesion molecule TAG-1/axonin-1, with dissociation constants of 0.3 nM and 0.04 nM, respectively. Phosphacan binding was decreased by approximately 70% following chondroitinase treatment, whereas binding of neurocan was not affected. The contribution of chondroitin sulfate chains to the binding of neurocan and phosphacan to TAG-1/axonin-1 is therefore the opposite of that previously observed for their binding to two other Ig-superfamily neural cell adhesion molecules, Ng-CAM/L1 and N-CAM. Moreover, whereas phosphacan interactions with certain proteins are mediated at least in part by N-linked oligosaccharides on the proteoglycan, N-deglycosylation of phosphacan had no effect on its binding to TAG-1/axonin-1. In addition to the chondroitin sulfate proteoglycans described above, we have demonstrated that N-CAM is a high-affinity ligand of TAG-1/axonin-1 (Kd approximately 1 nM), and specific binding of TAG-1/axonin-1 to tenascin-C was also observed (Kd approximately 9 nM). Immunocytochemical studies of embryonic and early postnatal nervous tissue showed an overlapping localization of TAG-1/axonin-1 with all four of these ligands, further supporting the biological significance of their ability to interact in vitro.

The neuronal chondroitin sulfate proteoglycan neurocan binds to the neural cell adhesion molecules Ng-CAM/L1/NILE and N-CAM, and inhibits neuronal adhesion and neurite outgrowth.

We have previously shown that aggregation of microbeads coated with N-CAM and Ng-CAM is inhibited by incubation with soluble neurocan, a chondroitin sulfate proteoglycan of brain, suggesting that neurocan binds to these cell adhesion molecules (Grumet, M., A. Flaccus, and R. U. Margolis. 1993. J. Cell Biol. 120:815). To investigate these interactions more directly, we have tested binding of soluble 125I-neurocan to microwells coated with different glycoproteins. Neurocan bound at high levels to Ng-CAM and N-CAM, but little or no binding was detected to myelin-associated glycoprotein, EGF receptor, fibronectin, laminin, and collagen IV. The binding to Ng-CAM and N-CAM was saturable and in each case Scatchard plots indicated a high affinity binding site with a dissociation constant of approximately 1 nM. Binding was significantly reduced after treatment of neurocan with chondroitinase, and free chondroitin sulfate inhibited binding of neurocan to Ng-CAM and N-CAM. These results indicate a role for chondroitin sulfate in this process, although the core glycoprotein also has binding activity. The COOH-terminal half of neurocan was shown to have binding properties essentially identical to those of the full-length proteoglycan. To study the potential biological functions of neurocan, its effects on neuronal adhesion and neurite growth were analyzed. When neurons were incubated on dishes coated with different combinations of neurocan and Ng-CAM, neuronal adhesion and neurite extension were inhibited. Experiments using anti-Ng-CAM antibodies as a substrate also indicate that neurocan has a direct inhibitory effect on neuronal adhesion and neurite growth. Immunoperoxidase staining of tissue sections showed that neurocan, Ng-CAM, and N-CAM are all present at highest concentration in the molecular layer and fiber tracts of developing cerebellum. The overlapping localization in vivo, the molecular binding studies, and the striking effects on neuronal adhesion and neurite growth support the view that neurocan may modulate neuronal adhesion and neurite growth during development by binding to neural cell adhesion molecules.