Activity-dependent remodeling of chondroitin sulfate proteoglycans extracellular matrix in the hypothalamo-neurohypophysial system.

The hypothalamo-neurohypophysial system (HNS) consisting of arginine vasopressin (AVP) and oxytocin (OXT) magnocellular neurons shows the structural plasticity including the rearrangement of synapses, dendrites, and neurovascular contacts during chronic physiological stimulation. In this study, we examined the remodeling of chondroitin sulfate proteoglycans (CSPGs), main extracellular matrix (ECM), in the HNS after salt loading known as a chronic stimulation to cause the structural plasticity. In the supraoptic nucleus (SON), confocal microscopic observation revealed that the immunoreactivity of 6B4 proteoglycans (PG) was observed mainly at AVP-positive magnocellular neurons but that of neurocan was seen chiefly at OXT-positive magnocellular neurons. The immunoreactivity of phosphacan and aggrecan was seen at both AVP- and OXT-positive magnocellular neurons. Electron microscopic observation further showed that the immunoreactivity of phosphacan and neurocan was observed at astrocytic processes to surround somata, dendrites, and terminals, but not synaptic junctions. In the neurohypophysis (NH), the immunoreactivity of phosphacan, 6B4 PGs, and neurocan was observed at AVP-positive magnocellular terminals, but the reactivity of Wisteria floribunda agglutinin lectin was seen at OXT-positive ones. The immunoreactivity of versican was found at microvessel and that of aggrecan was not detected in the NH. Quantitative morphometrical analysis showed that the chronic physiological stimulation by 7-day salt loading decreased the level of 6B4 PGs in the SON and the level of phosphacan, 6B4 PGs, and neurocan in the NH. These results suggest that the extracellular microenvironment of CSPGs is different between AVP and OXT magnocellular neurons and activity-dependent remodeling of CSPGs could be involved in the structural plasticity of the HNS.

Construction of perineuronal net-like structure by cortical neurons in culture.

Perineuronal nets consisting of chondroitin sulfate proteoglycans and hyaluronic acid are associated with distinct neuronal populations in mammalian brain. Whether neurons or glia cells produce these surface-associated chondroitin sulfate proteoglycan perineuronal nets has remained in question. In the present study, we observed perineuronal net-like structure by rat cortical neurons in dissociated culture using Wisteria floribunda aggulutinin, hyaluronic acid binding protein, and the antibodies recognizing chondroitin sulfate proteoglycans. The double labeling experiments showed that perineuronal net-like structure labeled with Wisteria floribunda aggulutinin was observed often at parvalbumin-positive neurons in dissociated cortical culture without glia. Perineuronal net-like structure was not seen at the early stage of culture, but they became visible concomitantly with neuronal maturation after longer culture. High magnification observation further demonstrated that Wisteria floribunda aggulutinin labeling on cortical neurons was seen as numerous puncta along surface of somata and proximal dendrites, but not axons and synapses. Perineuronal net-like structure on cultured neurons was also visualized using chondroitin sulfate proteoglycan-specific antibodies and hyaluronic acid binding protein. Double labeling study demonstrated that perineuronal net-like structure in cultured cortical neurons was composed of chondroitin sulfate proteoglycans such as neurocan and phosphacan. The hyaluronidase treatment of live neurons abolished cellular labeling of hyaluronic acid binding protein and concomitantly diminished that of Wisteria floribunda aggulutinin. These results indicate that cultured cortical neurons are able to construct perineuronal net-like structure without glial cells.

Neuronal expression of the chondroitin sulfate proteoglycans receptor-type protein-tyrosine phosphatase beta and phosphacan.

Receptor-type protein-tyrosine phosphatase beta (RPTPbeta) and its spliced variant phosphacan are major components of chondroitin sulfate proteoglycans in the CNS. In this study, expression and localization of RPTPbeta and phosphacan were examined in developing neurons by immunological analyses using 6B4, 3F8, and anti-PTP antibodies and reverse transcription-polymerase chain reaction (RT-PCR). Light microscopic immunohistochemistry showed that 6B4 RPTPbeta/phosphacan immunoreactivity was observed around neurons in the cortical plate. Further ultrastructural observation showed that 6B4 RPTPbeta/phosphacan immunoreactivity was observed mainly at the membrane of migrating neurons and radial glia. Immunocytochemical analysis revealed that RPTPbeta immunoreactivity was observed in cultured cerebral, hippocampal, and cerebellar neurons in addition to type-1 and type-2 astrocytes. Western analysis further demonstrated that the shorter receptor form of RPTPbeta (sRPTPbeta) was detected from cell lysate of cortical and hippocampal neurons using 6B4 and anti-PTP antibodies, while sRPTPbeta of cerebellar neurons and type-1 astrocytes was recognized only by anti-PTP antibody. Phosphacan was detected from neuronal culture supernatants of cortical, hippocampal, and cerebellar neurons, but not from type-1 astrocytes using 6B4 and 3F8 antibodies. RT-PCR analysis demonstrated the prominent expression of sRPTPbeta and phosphacan mRNAs in cortical neurons, and that of sRPTPbeta mRNA in type-1 astrocytes. During culture development of cortical neurons, the immunoreactivity of 6B4 sRPTPbeta was observed entirely on the neuronal surface including somata, dendrites, axons, and growth cones at earlier stages of cortical neuronal culture such as stages 2 and 3, while, after longer culture, 6B4 sRPTPbeta immunoreactivity in stages 4 and 5 neurons was detected at dendrites and somata and disappeared from axons, and was not observed over axonal terminals and postsynaptic spines. These results demonstrate that neurons are able to express sRPTPbeta on their cellular surface and to secrete phosphacan, and neuronal expression of sRPTPbeta may modulate neuronal differentiation including neuritogenesis and synaptogenesis.

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.

Developmentally regulated expression of brain-specific chondroitin sulfate proteoglycans, neurocan and phosphacan, in the postnatal rat hippocampus.

Developmental changes in the distribution of brain-specific chondroitin sulfate proteoglycans, neurocan and phosphacan/RPTPzeta/beta, in the hippocampus of the Sprague-Dawley rat were examined using monoclonal antibodies 1G2 and 6B4. The 1G2 immunoreactivity was predominant in the neonatal hippocampus while the 6B4 immunoreactivity was predominant in the mature hippocampus. Moderate 1G2 immunoreactivity was detected in the dentate gyrus and subiculum immediately after birth. Immunoreactivity reached a peak on postnatal days 7-10 (P7-P10) when intense 1G2 labeling was present throughout the neuropil layers of the hippocampus except the mossy fiber tract. 6B4 immunoreactivity was limited in the stratum lacunosum moleculare of CA1 in the neonatal hippocampus. It gradually increased by P21 when diffuse 6B4 immunoreactivity was detected in the stratum oriens and radiatum of Ammon's horn, and in the hilus and inner one-third molecular layer of the dentate gyrus, while 1G2 immunoreactivity decreased after P21. In the adult hippocampus, moderate 6B4 immunoreactivity was present in the stratum oriens and radiatum of Ammon's horn, and in the hilus and inner one-third molecular layer of the dentate gyrus, but not in the mossy fiber tract. In addition, strong 6B4 labeling appeared around a subset of neurons after P21. The results suggest that neurocan may have a role in the development of neuronal organization, while phosphacan/RPTPzeta/beta may contribute to the maintenance and plasticity of synaptic structure and function. Furthermore, the absence of 1G2 and 6B4 immunoreactivities in the stratum lucidum suggests that neurocan and phosphacan/RPTPzeta/beta may function as a barrier for the extension of mossy fibers and provide an environment permissive for fasciculation of the mossy fibers.

Upregulated expression of N-syndecan, a transmembrane heparan sulfate proteoglycan, in differentiated neural stem cells.

Adult rat hippocampus-derived neural stem cells are incorporated into neural tissues, and differentiate to neuronal and glial cells. However, the cell surface protein molecules are, to date, undefined. RT-PCR, immunoblotting and immunocytochemistry showed the increased expression of N-syndecan, a transmembrane heparan sulfate proteoglycan, in the neural stem cells after the differentiation induced by retinoic acid. Our data indicate that N-syndecan may be involved in the differentiation of neural stem cells.

Inhibitory effects of neurocan and phosphacan on neurite outgrowth from retinal ganglion cells in culture.

PURPOSE: Neurocan and phosphacan are nervous tissue-specific chondroitin sulfate proteoglycans (CSPGs) that are highly expressed in postnatal rat retina. To elucidate potential roles of neurocan and phosphacan on neurite outgrowth from retinal ganglion cells (RGCs), in vitro experiments were conducted with purified RGCs. METHODS: Neurocan and phosphacan were purified from postnatal rat brain by DEAE-column chromatography and subsequent gel chromatography. RGCs were obtained from postnatal rat retinas by a two-step immunopanning procedure using an anti-Thy 1,1 antibody and an anti-macrophage antibody. Neurite outgrowth from RGCs was examined on poly-L-lysine (PLL)-conditioned plates, and PLL-conditioned plates treated with neurocan or phosphacan. RESULTS: Compared with PLL-conditioned plates, neurocan and phosphacan inhibited neurite outgrowth from RGCs at 48 and 72 hours after seeding. When chondroitin sulfate side chains linked to the core proteins were digested by chondroitinase ABC, the inhibitory effect remained, indicating that the core proteins are related to the effect. Furthermore, the digestion of chondroitin sulfate side chains linked to phosphacan core protein significantly promoted the inhibitory effect of phosphacan on neurite outgrowth from RGCs. CONCLUSIONS: Neurocan and phosphacan, which are highly expressed in postnatal rat retina, inhibit neurite outgrowth from postnatal rat RGCs, indicating that these proteoglycans may be inhibitory factors against neurite outgrowth from RGCs during retinal development.

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.

Regulation of neuregulin expression in the injured rat brain and cultured astrocytes.

In this report, we investigated whether reactive astrocytes produce neuregulins (glial growth factor 2/heregulin/acetylcholine receptor-inducing activity or neu differentiation factor) and its putative receptors, ErbB2 and ErbB3 tyrosine kinases, in the injured CNS in vivo. Significant immunoreactivities with anti-neuregulin, anti-ErbB2, and anti-ErbB3 antibodies were detected on astrocytes at the injured site 4 d after injury to the adult rat cerebral cortex. To elucidate the mechanisms for the upregulation of neuregulin expression in astrocytes, primary cultured astrocytes were treated with certain reagents, including forskolin, that are known to elevate the intracellular level of cAMP and induce marked morphological changes in astrocytes. Western blot analysis showed that the expression of a 52 kDa membrane-spanning form of a neuregulin protein was enhanced in cultured astrocytes after administration of forskolin. The upregulation of glial fibrillary acidic protein was also observed in astrocytes treated with forskolin. In contrast, inactivation of protein kinase C because of chronic treatment with phorbol ester 12-O-tetradecanoyl phorbol 13-acetate downregulated the expression of the 52 kDa isoform, although other splice variants with apparent molecular sizes of 65 and 60 kDa were upregulated. These results suggest that the enhancement of neuregulin expression at injured sites is induced, at least in part, by elevation in intracellular cAMP levels and/or a protein kinase C signaling pathway. The neuregulin expressed on reactive astrocytes may stimulate their proliferation and support the survival of neurons surrounding cortical brain wounds in vivo.

Neuroglycan C, a neural tissue-specific transmembrane chondroitin sulfate proteoglycan, in retinal neural network formation.

PURPOSE: Neuroglycan C (NGC) is a transmembrane chondroitin sulfate proteoglycan present exclusively in central nervous system tissues. In the current study the expression pattern and characterization of NGC during the development of the retina were investigated. METHODS: Expressional changes of NGC mRNAs during rat retinal development were examined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). The localization and characterization of NGC core proteins were investigated by immunoblot analysis and immunohistochemistry using an anti-NGC antibody. RESULTS: Immunohistochemical analysis revealed that NGC was highly expressed in the nerve fiber layer (NFL) and inner plexiform layer (IPL) in rat postnatal developing retina. At embryonal stages, NGC immunoreactivities were faint. In contrast, at postnatal developmental stages (approximately postnatal day [P]7), intense immunoreactivity was observed in the NFL and IPL, where active dendrite branching was observed, and conventional synapses began to be formed. As retinal layer differentiation proceeded (from P14 to P42), immunoreactivities in the inner retinal layers gradually became fainter. Immunoblot and semiquantitative RT-PCR analyses showed that the peak level of NGC expression occurred on approximately P7 and P14. Glycosylation of the NGC core protein changed as the retinal layers matured. In immunoelectron microscopic analysis, NGC immunoreactivity was located on the axonal membranes of neuronal cells in the postnatal retina, whereas immunoreactivity was reduced on membranes at the adult stage. In retinal ganglion cells in vitro, NGC was highly localized in their spiny budding neurites. CONCLUSIONS: The results show spatiotemporal expression patterns of NGC, and suggest that it plays a role in the formation of neural networks in retinal development.

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.

Involvement of gangliosides in glycosylphosphatidylinositol-anchored neuronal cell adhesion molecule TAG-1 signaling in lipid rafts.

The association of ganglioside GD3 with TAG-1, a glycosylphosphatidylinositol-anchored neuronal cell adhesion molecule, was examined by coimmunoprecipitation experiments. Previously, we have shown that the anti-ganglioside GD3 antibody (R24) immunoprecipitated the Src family kinase Lyn from the rat cerebellum, and R24 treatment of primary cerebellar cultures induced Lyn activation and rapid tyrosine phosphorylation of an 80-kDa protein (p80). We now report that R24 coimmunoprecipitates a 135-kDa protein (p135) from primary cerebellar cultures. Treatment with phosphatidylinositol-specific phospholipase C revealed that p135 was glycosylphosphatidylinositol-anchored to the membrane. It was identified as TAG-1 by sequential immunoprecipitation with an anti-TAG-1 antibody. Antibody-mediated cross-linking of TAG-1 induced Lyn activation and rapid tyrosine phosphorylation of p80. Selective inhibitor for Src family kinases reduced the tyrosine phosphorylation of p80. Sucrose density gradient analysis revealed that the TAG-1 and tyrosine-phosphorylated p80 in cerebellar cultures were present in the lipid raft fraction. These data show that TAG-1 transduces signals via Lyn to p80 in the lipid rafts of the cerebellum. Furthermore, degradation of cell-surface glycosphingolipids by endoglycoceramidase induced an alteration of TAG-1 distribution on an OptiPrep gradient and reduced the TAG-1-mediated Lyn activation and tyrosine phosphorylation of p80. These observations suggest that glycosphingolipids are involved in TAG-1-mediated signaling in lipid rafts.

Comparative mapping of seven genes in mouse, rat and Chinese hamster chromosomes by fluorescence in situ hybridization.

By fluorescence in situ hybridization (FISH) using mouse probes, we assigned homologues for cathepsin E (Ctse), protocadherin 10 (Pcdh10, alias OL-protocadherin, Ol-pc), protocadherin 13 (Pcdh13, alias protocadherin 2c, Pcdh2c), neuroglycan C (Cspg5) and myosin X (Myo10) genes to rat chromosomes (RNO) 13q13, 2q24-->q25, 18p12-->p11, 8q32.1 and 2q22.1-->q22.3, respectively. Similarly, homologues for mouse Ctse, Pcdh13, Cspg5 and Myo10 genes and homologues for rat Smad2 (Madh2) and Smad4 (Madh4) genes were assigned to Chinese hamster chromosomes (CGR) 5q28, 2q17, 4q26, 2p29-->p27, 2q112-->q113 and 2q112-->q113, respectively. The chromosome assignments of homologues of Ctse and Cspg5 reinforced well-known homologous relationships among mouse chromosome (MMU) 1, RNO 13 and CGR 5q, and among MMU 9, RNO 8 and CGR 4q, respectively. The chromosome locations of homologues for Madh2, Madh4 and Pcdh13 genes suggested that inversion events were involved in chromosomal rearrangements in the differentiation of MMU 18 and RNO 18, whereas most of MMU 18 is conserved as a continuous segment in CGR 2q. Furthermore, the mapping result of Myo10 and homologues suggested an orthologous segment of MMU 15, RNO 2 and CGR 2.

Spatiotemporal expression patterns of 6B4 proteoglycan/phosphacan in the developing rat retina.

PURPOSE: To investigate expression of 6B4 proteoglycan/phosphacan, the major constituent of chondroitin sulfate proteoglycan and a possible modulator of neural network formation in the developing central nervous system, in developing rat retina. METHODS: Changes in expression and localization of 6B4 proteoglycan in developing rat retina were investigated by reverse transcription-initiated polymerase chain reaction (RT-PCR), immunohistochemistry, and immunoblot analysis. RESULTS: Semiquantitative RT-PCR revealed that mRNA expression of 6B4 proteoglycan in retinas peaked at postnatal day 14 (P14) and then decreased at P42. Immunohistochemical analyses using MAb 6B4, a monoclonal antibody against 6B4 proteoglycan, revealed faint immunoreactivity in the inner aspects of the retina at embryonal day 16 (E16). At birth, weak immunoreactivity was present in the nerve fiber layer (NFL) and inner plexiform layer (IPL). At P7 and P14, the NFL, IPL, and outer plexiform layer (OPL) stained intensely, but the ganglion cell layer (GCL) remained unstained. Between P21 and P42, immunoreactivity in the NFL and IPL weakened slightly. Immunoblot analyses showed a MAb 6B4 immunopositive band in the retinal soluble fraction treated with chondroitinase ABC. The amount of the immunopositive band increased rapidly as retinal development proceeded. Surprisingly, a significant amount of the immunopositive band was present in the retina even before digestion with chondroitinase ABC, indicating that at least part of 6B4 proteoglycan in rat retina exists in a non-proteoglycan form. CONCLUSIONS: The existence of 6B4 proteoglycan/phosphacan was thus demonstrated in rat retina, although some biochemical parameters were different from those of the 6B4 proteoglycan seen in brain.

Effects of lipid-derivatized glycosaminoglycans (GAGs), a novel probe for functional analyses of GAGs, on cell-to-substratum adhesion and neurite elongation in primary cultures of fetal rat hippocampal neurons.

The effects of glycosaminoglycans (GAG) on cell-to-substratum adhesion and neurite elongation were examined in primary cultures of fetal rat hippocampal neurons using tissue culture dishes coated with GAGs coupled to dipalmitoylphosphatidylethanolamine (PE), a novel probe for biological functions of GAGs. Both chondroitin sulfate conjugate to PE (CS-PE) and hyaluronic acid conjugate to PE (HA-PE) promoted neurite elongation from neurons in a dose-dependent manner when immobilized onto polylysine-coated dishes at various concentrations up to 1.0 microg/ml. The coating of CS-PE or HA-PE at a concentration higher than 1.0 microg/ml resulted in failure of neurite extension and adhesion of neurons to the substrata. In contrast, heparin conjugate to PE (HP-PE) did not exert any effects on neurite elongation or on cell attachment at these concentrations. These findings suggest that GAGs serve as a modulator for neurite elongation during neuronal network formation in the developing central nervous system.

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.

Genomic organization and expression pattern of mouse neuroglycan C in the cerebellar development.

Neuroglycan C (NGC) is a membrane-spanning chondroitin sulfate proteoglycan with an epidermal growth factor module that is expressed predominantly in the brain. Cloning studies with mouse NGC cDNA revealed the expression of three distinct isoforms (NGC-I, -II, and -III) in the brain and revealed that the major isoform showed 94. 3% homology with the rat counterpart. The NGC gene comprised six exons, was approximately 17 kilobases in size, and was assigned to mouse chromosome band 9F1 by fluorescence in situ hybridization. Western blot analysis demonstrated that, although NGC in the immature cerebellum existed in a proteoglycan form, most NGC in the mature cerebellum did not bear chondroitin sulfate chain(s), indicating that NGC is a typical part-time proteoglycan. Immunohistochemical studies showed that only the Purkinje cells were immunopositive in the cerebellum. In the immature Purkinje cells, NGC, probably the proteoglycan form, was immunolocalized to the soma and thick dendrites on which the climbing fibers formed synapses, not to the thin branches on which the parallel fibers formed synapses. This finding suggests the involvement of NGC in the differential adhesion and synaptogenesis of the climbing and parallel fibers with the Purkinje cell dendrites.

Molecular interactions of neural chondroitin sulfate proteoglycans in the brain development.

Aggrecan family proteoglycans, phosphacan/RPTPzeta/beta, and neuroglycan C (NGC) are the major classes of chondroitin sulfate proteoglycan in the developing mammalian brain. A multidomain is a common structural feature of these proteoglycans which can interact with various molecules including growth factors, cell adhesion molecules, and extracellular matrix molecules. Individual proteoglycans are distributed in the developing brain in a distinct temporal and spatial pattern, suggesting that they are involved in distinct phases of the brain development through multiple molecular interactions. This review mainly summarizes recent studies on the involvement of these three classes of proteoglycan in cell-cell and cell-substratum interactions during the brain development. Their expressions and proposed functional roles in injured brains are also mentioned. In addition, this review briefly covers potential functions of other neural chondroitin sulfate proteoglycans such as decorin, testican, NG2 proteoglycan, and amyloid precursor protein (APP) in developing and injured brains.