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PubMed1996

Structural and electron microscopic analysis of neurocan and recombinant neurocan fragments.

Neurocan, a nervous tissue-specific chondroitin sulfate proteoglycan of the aggrecan family which has been shown to interact with neural cell adhesion molecules and tenascin, could be visualized by rotary shadowing electron microscopy as two globular domains interconnected by an extended flexible filament of 60-90 nm. Several recombinant neurocan fragments generated in the human embryonic kidney cell line 293 represent as observed by electron microscopy the expected parts of this structure, which indicates a correct folding of these molecules. Biological activity of the recombinant N-terminal globular domain could be demonstrated by its coelution with hyaluronan in gel permeation chromatography. In addition, the modification of the recombinant fragments with certain carbohydrate structures was analyzed. High mannose oligosaccharides could be mapped to the N-terminal globular domain of the brain-derived molecule. Only recombinant fragments containing parts of the central region of the molecule were modified with chondroitin sulfate chains and with the HNK-1 epitope, and could be considerably altered in their migratory behavior on SDS-polyacrylamide gel electrophoresis by neuraminidase treatment. These findings and the electron microscopic shape indicate a mucin-like character for the central neurocan region.

Amino Acid Sequence↗

PubMed2004

Heparan sulphate proteoglycans interact with neurocan and promote neurite outgrowth from cerebellar granule cells.

We found that neurocan, a major brain chondroitin sulphate proteoglycan, interacts with HSPGs (heparan sulphate proteoglycans) such as syndecan-3 and glypican-1. Binding of these HSPGs to neurocan was prevented by treatment of the HSPGs with heparitinases I and II, but not by treatment of neurocan with chondroitinase ABC. Scatchard plot analysis indicated that neurocan has two binding sites for these HSPGs with different affinities. It is known that neurocan in the rodent brain is proteolytically processed with aging into N- and C-terminal fragments. When a mixture of whole neurocan and N- and C-terminal fragments prepared from neonatal mouse brains or recombinant N- and C-terminal fragments was applied to a heparin column, the whole molecule and both the N- and C-terminal fragments bound to heparin. A centrifugation cell adhesion assay indicated that both the N- and C-terminal neurocan fragments could interact with these HSPGs expressed on the cell surface. To examine the biological significance of the HSPG-neurocan interaction, cerebellar granule cells expressing these HSPGs were cultured on the recombinant neurocan substrate. A significant increase in the rate of neurite outgrowth was observed on the wells coated with the C-terminal neurocan fragment, but not with the N-terminal one. Neurite outgrowth-promoting activity was inhibited by pretreatment of neurocan substrate with heparin or the addition of heparitinase I to culture medium. These results suggest that HSPGs such as syndecan-3 and glypican-1 serve as the cell-surface receptor of neurocan, and that the interaction of these HSPGs with neurocan through its C-terminal domain is involved in the promotion of neurite outgrowth.

Animals↗

PubMed1999

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.

Animals↗

PubMed1998

Occurrence of a N-terminal proteolytic fragment of neurocan, not a C-terminal half, in a perineuronal net in the adult rat cerebrum.

Neurocan is a nervous tissue-unique chondroitin sulfate proteoglycan (CSPG) whose expression and proteolytic cleavage are developmentally regulated. In the adult rat brain, neurocan is completely cleaved into some proteoglycan fragments including the C-terminal half known as neurocan-C and a N-terminal fragment with a 130 kDa core glycoprotein (neurocan-130). We describe here the differential distribution of these two neurocan-derived CSPGs in the adult rat cerebrum and the occurrence of neurocan-130 as a new member of a perineuronal net-constituting molecule. At the light microscopic level, neurocan-130 exhibited pericellular localization around a subset of neurons in addition to diffuse distribution in the neuropil. In contrast, neurocan-C was distributed only diffusely in the neuropil. Double staining with anti-neurocan-130 and anti-synaptophysin antibodies suggested that neurocan-130 was localized in the vicinity of the synapses, but not at the synapses. Immunoelectron microscopy showed that neurocan-130 was mainly localized in the cytoplasm of glial cell processes, the so-called glial perineuronal net, encompassing the cell bodies of certain neurons. The presence of neurocan-130 in a limited number of glial cells may reflect some functional heterogeneity of the glia.

Age Factors↗

PubMed1994

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.

Aggrecans↗

PubMed2000

Neurocan is upregulated in injured brain and in cytokine-treated astrocytes.

Injury to the CNS results in the formation of the glial scar, a primarily astrocytic structure that represents an obstacle to regrowing axons. Chondroitin sulfate proteoglycans (CSPG) are greatly upregulated in the glial scar, and a large body of evidence suggests that these molecules are inhibitory to axon regeneration. We show that the CSPG neurocan, which is expressed in the CNS, exerts a repulsive effect on growing cerebellar axons. Expression of neurocan was examined in the normal and damaged CNS. Frozen sections labeled with anti-neurocan monoclonal antibodies 7 d after a unilateral knife lesion to the cerebral cortex revealed an upregulation of neurocan around the lesion. Western blot analysis of extracts prepared from injured and uninjured tissue also revealed substantially more neurocan in the injured CNS. Western blot analysis revealed neurocan and the processed forms neurocan-C and neurocan-130 to be present in the conditioned medium of highly purified rat astrocytes. The amount detected was increased by transforming growth factor beta and to a greater extent by epidermal growth factor and was decreased by platelet-derived growth factor and, to a lesser extent, by interferon gamma. O-2A lineage cells were also capable of synthesizing and processing neurocan. Immunocytochemistry revealed neurocan to be deposited on the substrate around and under astrocytes but not on the cells. Astrocytes therefore lack the means to retain neurocan at the cell surface. These findings raise the possibility that neurocan interferes with axonal regeneration after CNS injury.

Animals↗

PubMed2000

Dynamic spatiotemporal expression patterns of neurocan and phosphacan indicate diverse roles in the developing and adult mouse olfactory system.

The chondroitin sulfate proteoglycans neurocan and phosphacan are believed to modulate neurite outgrowth by binding to cell adhesion molecules, tenascin, and the differentiation factors heparin-binding growth-associated molecule and amphoterin. To assess the role of these chondroitin sulfate proteoglycans in the olfactory system, we describe here their expression patterns during both embryonic and postnatal development in the mouse. Immunoreactivity for neurocan was first detected in primary olfactory neurons at embryonic day 11. 5 (E11.5). Neurocan was expressed by primary olfactory axons as they extended toward the rostral pole of the telencephalon as well as by their arbors in glomeruli after they contacted the olfactory bulb. The role of neurocan was examined by growing olfactory neurons on an extracellular matrix substrate containing neurocan or on extracellular matrix in the presence of soluble neurocan. In both cases, neurocan strongly promoted neurite outgrowth. These results suggest that neurocan supports the growth of primary olfactory axons through the extracellular matrix as they project to the olfactory bulb during development. Phosphacan, unlike neurocan, was present within the mesenchyme surrounding the E11.5 and E12.5 nasal cavity. This expression decreased at E13.5, concomitant with a transient appearance of phosphacan in nerve fascicles. Within the embryonic olfactory bulb, phosphacan was localised to the external and internal plexiform layers. However, during early postnatal development phosphacan was concentrated in the glomerular layer. These results suggest that phosphacan may play a role in delineating the pathway of growing olfactory axons as well as defining the laminar organization of the bulb. Together, the spatiotemporal expression patterns of neurocan and phosphacan indicate that these chondroitin sulfate proteoglycans have diverse in situ roles, which are dependent on context-specific interactions with extracellular and cell adhesion molecules within the developing olfactory nerve pathway.

Age Factors↗

PubMed2002

Transient expression of juvenile-type neurocan by reactive astrocytes in adult rat brains injured by kainate-induced seizures as well as surgical incision.

Neurocan is one of the major chondroitin sulfate proteoglycans expressed in nervous tissues. The expression of neurocan is developmentally regulated, and full-length neurocan is detected in juvenile brains but not in adult brains. In the present study, we demonstrated by western blot analysis that full-length neurocan transiently appeared in adult rat hippocampus when it was lesioned by kainate-induced seizures. Immunohistochemical studies showed that neurocan was detected mainly around the CA1 region although the seizure resulted in neuronal cell degeneration in both the CA1 and CA3 regions of the hippocampus. Double-labeling for neurocan mRNA and glial fibrillary acidic protein demonstrated that many reactive astrocytes expressed neurocan mRNA. The re-expression of full-length neurocan was also observed in the surgically injured adult rat brain. In contrast, the expression of other nervous tissue chondroitin sulfate proteoglycans, such as phosphacan and neuroglycan C, was not intensified but rather was either reduced in the kainate-lesioned hippocampus or in the surgically injured cerebral cortex. These observations indicate that induction of neurocan expression by reactive astrocytes is a common phenomenon in the repair process of adult brain injury, and therefore, it can be postulated that juvenile-type neurocan plays some roles in brain repair.

Animals↗

PubMed1995

Distribution of a brain-specific proteoglycan, neurocan, and the corresponding mRNA during the formation of barrels in the rat somatosensory cortex.

Neurocan is a developmentally regulated chondroitin sulphate proteoglycan in the rat brain. In the present study, spatiotemporal patterns of expression of neurocan and the corresponding mRNA were examined in the developing cortical barrel field of the rat brain by using a monoclonal antibody that was highly specific to neurocan and a riboprobe for a portion of the mRNA. Immunohistochemical analysis revealed that neurocan was distributed throughout the cerebral cortex during early postnatal development but was excluded from the centres of cortical barrels at the time of entry and arborization of thalamocortical axons. At this developmental stage, expression of neurocan mRNA was shown by in situ hybridization to be down-regulated in the barrel centres. When a row of whisker follicles was laser-cauterized on postnatal day 1, the pattern of expression of neurocan was disturbed in the row of barrels that corresponded to the lesioned whisker follicles in the contralateral somatosensory cortex. From these observations, it appears that neuronal stimuli through early thalamocortical fibres from the sensory periphery cause reduced expression of neurocan mRNA in neurocan-producing cells in the presumptive barrel centres. Our findings also suggest that the pattern of distribution of neurocan in early postnatal barrel fields may be due mainly to the down-regulation of expression of neurocan mRNA.

Animals↗

PubMed2000

Upregulated expression of neurocan, a nervous tissue specific proteoglycan, in transient retinal ischemia.

PURPOSE: Neurocan, a nervous tissue-specific chondroitin sulfate proteoglycan synthesized primarily by neurons, is expressed abundantly in developing rat retina, whereas it is rarely expressed in adult rat retinas. This study investigated the reexpression of neurocan in a pathologic condition of adult rat retina. METHODS: Transient retinal ischemia was produced by occlusion of the retinal artery for 60 minutes. After transient retinal ischemia, neurocan expression was investigated by reverse transcription-initiated polymerase chain reaction (RT-PCR), immunohistochemistry, and immunoblot analysis. RESULTS: Semiquantitative analysis using RT-PCR revealed that mRNA expression for neurocan increased at 24 hours after reperfusion. Furthermore, on immunoblot analysis using an anti-neurocan antibody, MAb 1G2, the intensity of the 220-kDa band as well as the 150-kDa band increased markedly at 24 and 72 hours after reperfusion. The 220-kDa band was predominant at 24 hours after reperfusion, whereas the intensity of the 150-kDa band became almost the same as that of the 220-kDa band at 72 hours after reperfusion. Immunohistochemical analysis revealed that upregulated neurocan immunoreactivity was associated with glial Müller cells. CONCLUSIONS: Thus, upregulated expression of neurocan in transient retinal ischemia was demonstrated. Furthermore, the immunohistochemical analysis revealed that the upregulated expression of neurocan is derived from Müller cells, although it has been thought that neurocan is synthesized by neurons so far. The neurocan expression by Müller cells suggests that this proteoglycan plays a role in the damage and repair processes in diseased retina.

Animals↗

PubMed1997

Immunohistochemical localization of neurocan and L1 in the formation of thalamocortical pathway of developing rats.

We used immunohistochemistry to examine possible molecular interactions between the subplate and growing thalamocortical axons in rat fetuses. In the cortical anlage of embryonic day 16 (E16), the subplate first appeared below the cortical plate. Among chondroitin sulfate proteoglycans, phosphacan was uniformly distributed throughout the cortical wall, whereas neurocan was localized only in the subplate at E16. Neural cell adhesion molecules, NCAM-H, TAG-1, and L1, were detected in the cortical anlage. Both cortical neurons and growing axons were diffusely immunopositive for NCAM-H, and TAG-1 immunoreactivity was found on immature neurons and cortical efferent axons but not on thalamocortical axons. L1 immunoreactivity was specifically localized on the growing thalamocortical axons. When the locations of neurocan and L1 were compared in the developing cortex, L1-bearing axons were found to extend to neurocan-immunopositive regions; neurocan immunoreactivity was intense in the subplate at E16, when small numbers of L1-immunoreactive thalamocortical axons began to invade the cortex. At E17, many L1-positive axons were observed in the subplate that expressed neurocan specifically. Double immunostaining showed that L1-positive axons and neurocan immunoreactivity overlapped in the subplate at E17. After E18, neurocan expression gradually extended to the lower part of the cortical plate; it extended to the entire cortex by E21, 1 day before birth. By E21, L1-bearing axons had invaded the lower part of the cortical plate. The present study demonstrated that the neurocan expression precedes growth of L1-bearing thalamocortical afferent fibers. Because neurocan can bind to L1 molecule in vitro, these results suggest that neurocan and L1 play some important roles in pathfinding of the thalamocortical afferent fibers during rat corticogenesis.

Animals↗

PubMed2003

Neurocan in the embryonic avian heart and vasculature.

The chondroitin sulfate proteoglycan (CSPG) neurocan was previously considered to be nervous-system specific. However, we have found neurocan in the embryonic heart and vasculature. In stage 11 quail embryos, neurocan was prominently expressed in the myocardium, dorsal mesocardium, heart-forming fields, splanchnic mesoderm, and vicinity of the extraembryonic vaculature, and at lower levels in the endocardium. A comparison of neurocan staining with QH1 staining of vascular endothelial cells demonstrates that neurocan is frequently expressed by cells adjacent to endothelial cells, rather than by endothelial cells themselves. In some cases, a dispersed subset of cells are neurocan-positive in a field of cells that otherwise appear uniform in morphology. Later in development, neurocan expression becomes relatively limited to the nervous system. However, even in 10-day embryos, neurocan is expressed in the chorio-allantoic membrane in the tissue that separates closely packed, small-diameter blood vessels. In summary, our results suggest that neurocan may function as a barrier that regulates vascular patterning during development.

Animals↗

PubMed1994

Interactions with tenascin and differential effects on cell adhesion of neurocan and phosphacan, two major chondroitin sulfate proteoglycans of nervous tissue.

We have studied interactions of tenascin with two chondroitin sulfate proteoglycans, neurocan and phosphacan. Neurocan is a multi-domain proteoglycan with a 136-kDa core protein that is synthesized by neurons and binds to hyaluronic acid, whereas the 173-kDa core protein of phosphacan, which is synthesized by glia, represents an extracellular variant of the receptor-type protein tyrosine phosphatase RPTP zeta/beta. Keratan sulfate-containing glycoforms of phosphacan (designated phosphacan-KS) are also present in brain. Immunocytochemical studies of early postnatal rat cerebellum demonstrated that the localization of neurocan, phosphacan, and phosphacan-KS all overlap extensively with that of tenascin, an extracellular matrix protein that modulates cell adhesion and migration. Binding studies using purified proteins covalently attached to fluorescent microbeads demonstrated that proteoglycan-coated beads co-aggregated with differently fluorescing beads coated with tenascin. The co-aggregation was specifically inhibited by Fab' fragments of antibodies against tenascin or the proteoglycans and by soluble neurocan, phosphacan, and tenascin. A solid phase radioligand binding assay confirmed that neurocan, phosphacan, and phosphacan-KS bind to tenascin but not to laminin and fibronectin. Chondroitinase treatment of the proteoglycans or addition of free chondroitin sulfate had no significant effect, indicating that the binding activity is mediated largely via the core glycoproteins. Scatchard analysis demonstrated high affinity binding of 125I-phosphacan, phosphacan-KS, and neurocan to a single site in tenascin, and neurocan and various glycoforms of phosphacan all inhibited binding of 125I-phosphacan to tenascin. In studies of cell adhesion to proteins adsorbed to Petri dishes, phosphacan inhibited adhesion of C6 glioma cells to tenascin whereas neurocan had no effect. Our results suggest that tenascin binds phosphacan and neurocan in vivo and that interactions between chondroitin sulfate proteoglycans and tenascin may play important roles in nervous tissue histogenesis, possibly by modulating signal transduction across the plasma membrane.

Animals↗

PubMed2006

Neuronal hyperactivity induces astrocytic expression of neurocan in the adult rat hippocampus.

Extracellular matrix molecules are involved in the cellular functions of proliferation, migration, morphological differentiation, and synaptic plasticity. One candidate molecule of the extracellular matrix is the chondroitin sulfate proteoglycan neurocan. To determine whether neurocan expression is regulated by neuronal activity in the adult rat brain, we studied changes in hippocampal neurocan mRNA and protein expression following electrical stimulation of the perforant pathway in urethane-anesthetized rats. After 24 h of intermittent, unilateral 20 Hz stimulation, in situ hybridization revealed increased neurocan mRNA in glial fibrillary acidic protein (GFAP)-positive astrocytes bilaterally in all hippocampal subfields. These changes were quantified in the dentate molecular layer, the termination zone of the perforant pathway, using laser microdissection in combination with quantitative reverse transcription-polymerase chain reaction (RT-PCR). Immediately after 24 h stimulation, a six-fold upregulation was detected, which returned to control levels by 3 days post-stimulation. Neurocan immunoreactivity was similarly upregulated bilaterally. Immunostaining intensity reached a maximum by 4 days and returned to control levels by 14 days. The pattern of neurocan expression in the hippocampus depended on the intensity and duration of electrical stimulation. Under conditions of less intense afferent stimulation (4-24 h of 2.0 Hz paired-pulse stimulation, interpulse interval 40 ms), increases in neurocan mRNA and immunoreactivity were restricted to the ipsilateral termination zone of the stimulated perforant pathway. This layer-specific neurocan upregulation was not affected by intraperitoneal application of the NMDA-receptor antagonist MK-801. In conclusion, our data indicate that synaptic activity regulates the astrocytic expression of neurocan in a graded manner.

Animals↗

PubMed2001

Expression of brain specific chondroitin sulfate proteoglycans, neurocan and phosphacan, in the developing and adult hippocampus of Ihara's epileptic rats.

Ihara's epileptic rats (IER) is an animal model of temporal lobe epilepsy with mycrodysgenesis, that exhibit abnormal migration of hippocampal neurons and recurrent spontaneous seizures. As an attempt to elucidate the roles of extracellular matrix molecules in the epileptogenecity and mossy fiber sprouting, immunohistochemical localization of brain specific chondroitin sulfate proteoglycans (CSPGs), neurocan and phosphacan, was examined in the hippocampus of postnatal IER and Sprague-Dawley (SD) rats using monoclonal antibodies 1G2 against neurocan and 6B4 against phosphacan. There was no difference in the expression of these two CSPGs between IER and SD rats in the 1st postnatal week. However, the expression of neurocan was poor in the hippocampus of IER in the 2nd and 3rd weeks whereas intense labeling of neurocan was present throughout the hippocampus of SD rats. Labeling of neurocan was almost absent in the hippocampus, while phosphacan was diffusely expressed in the stratum oriens and radiatum of Ammon's horn, and in the hilus and inner one-third molecular layer of the dentate gyrus at the 2nd month after birth. There was no difference in the expression of neurocan and phosphacan between IER and SD rats at the 2nd month after birth. By contrast, phosphacan was reduced in the inner molecular layer of the dentate gyrus in 8-month-old IER, while neurocan was reexpressed in the outer molecular layer and hilus in 3- and 8-month-old IER. It was suggested that the insufficient expression of neurocan may affect the development of neuronal organization in the hippocampus, and that the remodeling of extracellular matrix in the dentate gyrus may contribute to the mossy fiber sprouting into the inner molecular layer.

Aging↗

PubMed2003

Kainic acid-induced convulsions cause prolonged changes in the chondroitin sulfate proteoglycans neurocan and phosphacan in the limbic structures.

Systemic administration of kainic acid induces repeated convulsive seizures (KA convulsions) that result in neuropathological changes similar to temporal lobe epilepsy and the appearance of spontaneous recurrent seizures (SRS). The appearance of SRS is considered a result of the remodeling of neuronal networks following neuronal degeneration. We investigated the changes in chondroitin sulfate proteoglycans (CSPGs) in the limbic structures after KA convulsions in the rat using monoclonal antibodies 1G2, which recognizes full-length neurocan and the C-terminal half of neurocan, neurocan C, and 6B4, which recognize phosphacan and protein tyrosine phosphatase zeta. After KA convulsions, full-length neurocan appeared by 24 h and reached a peak by 48 to 72 h, whereas phosphacan decreased within 24 h in the hippocampus. In immunohistochemistry, neurocan increased in the limbic structures coincident with the appearance of reactive astrocytes. Phosphacan decreased coincident with pyramidal cell loss in the hippocampus, and the number of phosphacan-positive perineuronal nets around parvalbumin neurons decreased, whereas parvalbumin neurons were relatively conserved. In contrast, phosphacan increased in the entorhinal and piriform cortices in correlation with the severity of neuronal loss. Both neurocan and phosphacan recovered to the control level by 8 weeks after KA convulsions in some rats, but the changes in neurocan and phosphacan described above still persisted in more than half the rats. The results indicate that KA convulsions induce prolonged changes in neurocan and phosphacan similar to those in the developing rat brain and suggest a role of these CSPGs in the remodeling of neuronal networks related to the establishment or enhancement of epileptogenesis.

Animals↗

PubMed1996

Analysis of neurocan structures interacting with the neural cell adhesion molecule N-CAM.

Neurocan is a brain-specific chondroitin sulfate proteoglycan, which has been shown to bind to the neural cell adhesion molecule N-CAM and to inhibit its homophilic interaction. To study in more detail the structures of neurocan responsible for this interaction, various recombinant neurocan fragments were generated. The ability of these fragments to interact with N-CAM was investigated in several different in vitro assay systems, enzyme-linked immunosorbent assay-type binding assays, Covasphere-aggregation assays, and assays based on an optical biosensor (BIAcoreTM) system. The analysis of the homophilic N-CAM interaction in the BIAcore system revealed a KD of 64 nM. This homophilic interaction could be reduced by preincubation of soluble N-CAM with neurocan. Direct binding of N-CAM to immobilized neurocan core protein and recombinant neurocan fragments could also be demonstrated, and KD values between 25 and 100 nM were obtained. In addition, direct binding of N-CAM to chondroitin sulfate could be demonstrated. Binding of N-CAM to the immobilized neurocan core protein could be inhibited with all recombinant fragments containing chondroitin sulfate or major parts of the mucin-like central region of neurocan. For the inhibition of homophilic N-CAM interactions, however, a combination of globular and extended structures was required.

Animals↗

PubMed1996

Neurocan and phosphacan: two major nervous tissue-specific chondroitin sulfate proteoglycans.

Neurocan is a multidomain hyaluronan-binding chondroitin sulfate proteoglycan that is synthesized by neurons, whereas the astroglial proteoglycan phosphacan is an mRNA splice variant representing the entire extracellular portion of a receptor-type protein tyrosine phosphatase. A glycoform of phosphocan (phosphocan-KS) that contains both chondroitin sulfate and keratan sulfate is present in the postnatal rat central nervous system (CNS). The concentration of neurocan in brain increases during late embryonic development but then declines steeply during the early postnatal period together with hyaluronan, and neurocan also undergoes extensive proteolytic processing during the course of brain development. In contrast, the concentrations of both phosphocan and phosphocan-KS rise steadily after embryonic day 20 to reach a plateau at about 2 weeks postnatally. In the embryonic CNS the distribution of neurocan mRNA is more widespread than that of phosphocan, which is primarily present in regions of active cell proliferation. Neurocan mRNA is also present in areas where the proteoglycan is not expressed, and there is evidence that the short open reading frame in its 5'-leader may function as a cis-acting regulatory signal for the modulation of neurocan expression in the developing CNS. Neurocan and phosphocan bind saturably, reversibly, and with high affinity to neural cell adhesion molecules (Ng-CAM/L1, NCAM, TAG-1/axonin-1) and to tenascin-C. The proteoglycans and their ligands have overlapping localizations in the CNS, and binding of phosphocan to Ng-CAM/L1, NCAM, and tenascin-C is mediated by complex-type N-linked oligosaccharides on the proteoglycan. Neurocan and phosphocan also bind to neurons and are potent inhibitors of neuronal and glial adhesion and neurite outgrowth. Through their interactions with neural cell adhesion and extracellular matrix molecules, these proteoglycans may play a major role in modulating cell adhesion, neurite growth, and signal transduction across the plasma membrane during the development of the CNS.

Aging↗