Functional characterization of chondroitin sulfate proteoglycans of brain: interactions with neurons and neural cell adhesion molecules.

Ng-CAM and N-CAM are cell adhesion molecules (CAMs), and each CAM can bind homophilically as demonstrated by the ability of CAM-coated beads (Covaspheres) to self-aggregate. We have found that the extent of aggregation of Covaspheres coated with either Ng-CAM or N-CAM was strongly inhibited by the intact 1D1 and 3F8 chondroitin sulfate proteoglycans of rat brain, and by the core glycoproteins resulting from chondroitinase treatment of the proteoglycans. Much higher concentrations of rat chondrosarcoma chondroitin sulfate proteoglycan (aggrecan) core proteins had no significant effect in these assays. The 1D1 and 3F8 proteoglycans also inhibited binding of neurons to Ng-CAM when mixtures of these proteins were adsorbed to polystyrene dishes. Direct binding of neurons to the proteoglycan core glycoproteins from brain but not from chondrosarcoma was demonstrated using an assay in which cell-substrate contact was initiated by centrifugation, and neuronal binding to the 1D1 proteoglycans was specifically inhibited by the 1D1 monoclonal antibody. Different forms of the 1D1 proteoglycan have been identified in developing and adult brain. The early postnatal form (neurocan) was found to bind neurons more effectively than the adult proteoglycan, which represents the C-terminal half of the larger neurocan core protein. Our results therefore indicate that certain brain proteoglycans can bind to neurons, and that Ng-CAM and N-CAM may be heterophilic ligands for neurocan and the 3F8 proteoglycan. The ability of these brain proteoglycans to inhibit adhesion of cells to CAMs may be one mechanism to modulate cell adhesion and migration in the nervous system.

Immunoelectron microscopic localization of hyaluronic acid-binding region and link protein epitopes in brain.

The 1C6 monoclonal antibody to the hyaluronic acid-binding region weakly stained a 65-kD component in immunoblots of the chondroitin sulfate proteoglycans of brain, and the 8A4 monoclonal antibody, which recognizes two epitopes in the polypeptide portion of link protein, produced strong staining of a 45-kD component present in the brain proteoglycans. These antibodies were utilized to examine the localization of hyaluronic acid-binding region and link protein epitopes in rat cerebellum. Like the chondroitin sulfate proteoglycans themselves and hyaluronic acid, hyaluronic acid-binding region and link protein immunoreactivity changed from a predominantly extracellular to an intracellular (cytoplasmic and intra-axonal) location during the first postnatal month of brain development. The cell types which showed staining of hyaluronic acid-binding region and link protein, such as granule cells and their axons (the parallel fibers), astrocytes, and certain myelinated fibers, were generally the same as those previously found to contain chondroitin sulfate proteoglycans and hyaluronic acid. Prominent staining of some cell nuclei was also observed. In agreement with earlier conclusions concerning the localization of hyaluronic acid and chondroitin sulfate proteoglycans, there was no intracellular staining of Purkinje cells or nerve endings or staining of certain other structures, such as oligodendroglia and synaptic vesicles. The similar localizations and coordinate developmental changes of chondroitin sulfate proteoglycans, hyaluronic acid, hyaluronic acid-binding region, and link protein add further support to previous evidence for the unusual cytoplasmic localization of these proteoglycans in mature brain. Our results also suggest that much of the chondroitin sulfate proteoglycan of brain may exist in the form of aggregates with hyaluronic acid.

Immunocytochemical localization of a chondroitin sulfate proteoglycan in nervous tissue. II. Studies in developing brain.

In contrast to the intracellular (cytoplasmic) localization of chondroitin sulfate proteoglycans in adult brain (Aquino, D. A., R. U. Margolis, and R. K. Margolis, 1984, J. Cell Biol. 99:940-952), immunoelectron microscopic studies in immature (7 d postnatal) rat cerebellum demonstrated almost exclusively extracellular staining in the granule cell and molecular layers. Staining was also extracellular and/or associated with plasma membranes in the region of the presumptive white matter. Axons, which are unmyelinated at this age, generally did not stain, although faint intracellular staining was present in some astrocytes. At 10 and 14 d postnatal there was a significant decrease in extracellular space and staining, and by 21 d distinct cytoplasmic staining of neurons and astrocytes appeared. This intracellular staining further increased by 33 d so as to closely resemble the pattern seen in adult brain. Analyses of the proteoglycans isolated from 7-d-old and adult brain demonstrated that they have essentially identical biochemical compositions, immunochemical reactivity, size, charge, and density. These findings indicate that the antibodies used in this study recognize the same macromolecule in both early postnatal and adult brain, and that the localization of this proteoglycan changes progressively from an extracellular to an intracellular location during brain development.

Immunocytochemical localization of a chondroitin sulfate proteoglycan in nervous tissue. I. Adult brain, retina, and peripheral nerve.

Monospecific antibodies were prepared to a previously characterized chondroitin sulfate proteoglycan of brain and used in conjunction with the peroxidase-antiperoxidase technique to localize the proteoglycan by immunoelectron microscopy. The proteoglycan was found to be exclusively intracellular in adult cerebellum, cerebrum, brain stem, and spinal cord. Some neurons and astrocytes (including Golgi epithelial cells and Bergmann fibers) showed strong cytoplasmic staining. Although in the central nervous system there was heavy axoplasmic staining of many myelinated and unmyelinated fibers, not all axons stained. Staining was also seen in retinal neurons and glia (ganglion cells, horizontal cells, and Müller cells), but several central nervous tissue elements were consistently unstained, including Purkinje cells, oligodendrocytes, myelin, optic nerve axons, nerve endings, and synaptic vesicles. In sympathetic ganglion and peripheral nerve there was no staining of neuronal cell bodies, axons, myelin, or Schwann cells, but in sciatic nerve the Schwann cell basal lamina was stained, as was the extracellular matrix surrounding collagen fibrils. Staining was also observed in connective tissue surrounding the trachea and in the lacunae of tracheal hyaline cartilage. These findings are consistent with immunochemical studies demonstrating that antibodies to the chondroitin sulfate proteoglycan of brain also cross-react to various degrees with certain connective tissue proteoglycans.

Molecular organization of prolactin granules. II. Characterization of glycosaminoglycans and glycoproteins of the bovine prolactin matrix.

Prolactin (PRL) granules can be isolated from the anterior pituitary gland of adult cows in nearly 50% yield by use of a procedure previously developed for the fractionation of the rat pituitary. Treatment of the isolated bovine granules with 0.2% Lubrol PX results in the solubilization of most membranes present in the fractin but has only a limited effect on the matrices, which remain aggregated and can be recovered and purified by gradient centrifugation. These membraneless PRL granules, studied in detail by morphological and biochemical techniques, were found to contain only small amounts of contaminants (primarily growth hormone granules and small membrane fragments). SDS polyacrylamide gel electrophoresis revealed that, in comparison with other fractions isolated from the bovine pituitary, the membraneless granules have a simpler polypeptide composition including PRL (approximately 85%), growth hormone (approximately 8%), as well as approximately 13 minor bands with apparent mol wt ranging from 80,000 go 45,000. Many of these minor bands are accounted for by glycoproteins, as revealed by their binding of 125I-concanavalin A, and two of these are also stained blue by the stains-all procedure, a reaction specific for acidic glycoconjugates. Chemical analyses of the membraneless granule fractin revealed the presence of a heterogeneous mixture of complex carbohydrates. Among glycosaminoglycans, the major component is heparan sulfate, while hyaluronic acid and chondroitin sulfate ar present in smaller amounts. Moreover, some of the glycoproteins are sulfated and account for over 50% of the nondialyzable 35S radioactivity found in the fraction isolated from labeled slices. Although the concentration of glycosaminoglycans and glycoproteins is relatively low in membraneless granules, the possibility that their presence in the fraction is largely due to cross-contamination and/or artifactual adsorption could be excluded on two grounds. These are: (a) electron microscope radiautography of preparations obtained from [35S]sulfate- and D-[6-3H]glucosamine-labeled slices showed a significant labeling of PRL granules in both intact cells and membraneless granule pellets, and (b) a mixing experiment showed that membraneless granules contain very little macromolecular sulfate radiactivity adsorbed from the soluble glycoconjugates present in the pituitary homogenate.

Molecular organization of prolactin granules. III. Intracellular transport of sulfated glycosaminoglycans and glycoproteins of the bovine prolactin granule matrix.

The intracellular transport of sulfated glycosaminoglycans (heparan sulfate and chondroitin sulfate) and glycoproteins of the prolactin (PRL) granule matrix, as well as that of PRL, was studied using a system of double-labeled bovine anterior pituitary slices. [(35)S]sulfate was used to label sulfated macromolecules and L-[(3)H]leucine to label PRL. In membraneless granules (isolated from a PRL granule fraction after solubilization of the membrane with Lubrol PX), sulfated glycosaminoglycans and glycoproteins were considerably labeled after a 15- min pulse, while the hormone was still unlabeled. During the chase incubation, the specific radioactivity of granule PRL and the various complex carbohydrate classes first increased, reaching a peak after approximately 40 min, and then began to decline. After 4 h of chase incubation the radioactivity remaining in granule PRL and sulfated complex carbohydrates was 50-60 percent of that observed at 40 min. Thus, in pituitary mammotrophs a pool of sulfated glycoproteins and glycosaminoglycans is transported intracellularly in parallel with PRL. This finding corroborates the previous conclusion (Zanini et al., 1980 J. Cell. Biol. 86:260-272) that sulfated macromolecules are structural components of the granule matrix. The discharge of labeled PRL and complex carbohydrates from the slices to the incubation medium was also investigated. [(35)S]-glycosaminoglycans and glycoproteins were released at a rapid rate during the first 30-40 min of chase incubation, when PRL granules had not yet attained maximum specific activities. By 40 min, their release tended to level off but the radioactivity accumulating in the incubation medium was still much larger (approximately a fourfold increase) than the losses observed concomitantly in PRL granules. These discharge kinetics contrast with that of [(3)H]PRL, which was not released during the 1st h of chase incubation but then began to accumulate at a high rate in the medium, in parallel with its decrease in granules. Dopamin (5 x 10(-7) M) strongly inhibited the release of labeled PRL but had no detectable effect on the release of labeled glycosaminoglycans and glycoproteins or on the discharge of (35)S-macromolecules as revealed by SDS polyacrylamide gel electrophoresis of incubation media. Thus the releases of PRL and sulfated macromolecules have different kinetics and can be dissociated from each other. These data indicate that much of the flycosaminoglycans and glycoproteins release form pituitary slices originates from sites other than PRL granules, and that at least part of the complex carbohydrates of the PRL granule matrix might not be released with the hormone but rather remains associated with the mammotroph cells after exocytosis.

Light and electron microscopic studies on the localization of hyaluronic acid in developing rat cerebellum.

The hyaluronic acid-binding region was prepared by trypsin digestion of chondroitin sulfate proteoglycan aggregate from the Swarm rat chondrosarcoma, and biotinylated in the presence of hyaluronic acid and link protein. After isolation by gel filtration and HPLC in 4 M guanidine HCl, the biotinylated hyaluronic acid-binding region was used, in conjunction with avidin-peroxidase, as a specific probe for the light and electron microscopic localization of hyaluronic acid in developing and mature rat cerebellum. At 1 w postnatal, there is strong staining of extracellular hyaluronic acid in the presumptive white matter, in the internal granule cell layer, and as a dense band at the base of the molecular layer, surrounding the parallel fibers. This staining moves progressively towards the pial surface during the second postnatal week, and extracellular staining remains predominant through postnatal week three. In adult brain, there is no significant extracellular staining of hyaluronic acid, which is most apparent in the granule cell cytoplasm, and intra-axonally in parallel fibers and some myelinated axons. The white matter is also unstained in adult brain, and no staining was seen in Purkinje cell bodies or dendrites at any age. The localization of hyaluronic acid and its developmental changes are very similar to that previously found in immunocytochemical studies of the chondroitin sulfate proteoglycan in nervous tissue (Aquino, D. A., R. U. Margolis, and R. K. Margolis. 1984. J. Cell Biol. 99:1117-1129; Aquino, D. A., R. U. Margolis, and R. K. Margolis. J. Cell Biol. 99:1130-1139), and to recent results from studies using monoclonal antibodies to the hyaluronic acid-binding region and link protein. The presence of brain hyaluronic acid in the form of aggregates with chondroitin sulfate proteoglycans would be consistent with their similar localizations and coordinate developmental changes.

Glypican and biglycan in the nuclei of neurons and glioma cells: presence of functional nuclear localization signals and dynamic changes in glypican during the cell cycle.

We have investigated the expression patterns and subcellular localization in nervous tissue of glypican, a major glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan that is predominantly synthesized by neurons, and of biglycan, a small, leucine-rich chondroitin sulfate proteoglycan. By laser scanning confocal microscopy of rat central nervous tissue and C6 glioma cells, we found that a significant portion of the glypican and biglycan immunoreactivity colocalized with nuclear staining by propidium iodide and was also seen in isolated nuclei. In certain regions, staining was selective, insofar as glypican and biglycan immunoreactivity in the nucleus was seen predominantly in a subpopulation of large spinal cord neurons. The amino acid sequences of both proteoglycans contain potential nuclear localization signals, and these were demonstrated to be functional based on their ability to target beta-galactosidase fusion proteins to the nuclei of transfected 293 cells. Nuclear localization of glypican beta-galactosidase or Fc fusion proteins in transfected 293 cells and C6 glioma cells was greatly reduced or abolished after mutation of the basic amino acids or deletion of the sequence containing the nuclear localization signal, and no nuclear staining was seen in the case of heparan sulfate and chondroitin sulfate proteoglycans that do not possess a nuclear localization signal, such as syndecan-3 or decorin (which is closely related in structure to biglycan). Transfection of COS-1 cells with an epitope-tagged glypican cDNA demonstrated transport of the full-length proteoglycan to the nucleus, and there are also dynamic changes in the pattern of glypican immunoreactivity in the nucleus of C6 cells both during cell division and correlated with different phases of the cell cycle. Our data therefore suggest that in certain cells and central nervous system regions, glypican and biglycan may be involved in the regulation of cell division and survival by directly participating in nuclear processes.

Interactions of the chondroitin sulfate proteoglycan phosphacan, the extracellular domain of a receptor-type protein tyrosine phosphatase, with neurons, glia, and neural cell adhesion molecules.

Phosphacan is a chondroitin sulfate proteoglycan produced by glial cells in the central nervous system, and represents the extracellular domain of a receptor-type protein tyrosine phosphatase (RPTP zeta/beta). We previously demonstrated that soluble phosphacan inhibited the aggregation of microbeads coated with N-CAM or Ng-CAM, and have now found that soluble 125I-phosphacan bound reversibly to these neural cell adhesion molecules, but not to a number of other cell surface and extracellular matrix proteins. The binding was saturable, and Scatchard plots indicated a single high affinity binding site with a Kd of approximately 0.1 nM. Binding was reduced by approximately 15% after chondroitinase treatment, and free chondroitin sulfate was only moderately inhibitory, indicating that the phosphacan core glycoprotein accounts for most of the binding activity. Immunocytochemical studies of embryonic rat spinal phosphacan, Ng-CAM, and N-CAM have overlapping distributions. When dissociated neurons were incubated on dishes coated with combinations of phosphacan and Ng-CAM, neuronal adhesion and neurite growth were inhibited. 125I-phosphacan bound to neurons, and the binding was inhibited by antibodies against Ng-CAM and N-CAM, suggesting that these CAMs are major receptors for phosphacan on neurons. C6 glioma cells, which express phosphacan, adhered to dishes coated with Ng-CAM, and low concentrations of phosphacan inhibited adhesion to Ng-CAM but not to laminin and fibronectin. Our studies suggest that by binding to neural cell adhesion molecules, and possibly also by competing for ligands of the transmembrane phosphatase, phosphacan may play a major role in modulating neuronal and glial adhesion, neurite growth, and signal transduction during the development of the central nervous system.

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.

The tissue plasminogen activator (tPA)/plasmin extracellular proteolytic system regulates seizure-induced hippocampal mossy fiber outgrowth through a proteoglycan substrate.

Short seizure episodes are associated with remodeling of neuronal connections. One region where such reorganization occurs is the hippocampus, and in particular, the mossy fiber pathway. Using genetic and pharmacological approaches, we show here a critical role in vivo for tissue plasminogen activator (tPA), an extracellular protease that converts plasminogen to plasmin, to induce mossy fiber sprouting. We identify DSD-1-PG/phosphacan, an extracellular matrix component associated with neurite reorganization, as a physiological target of plasmin. Mice lacking tPA displayed decreased mossy fiber outgrowth and an aberrant band at the border of the supragranular region of the dentate gyrus that coincides with the deposition of unprocessed DSD-1-PG/phosphacan and excessive Timm-positive, mossy fiber termini. Plasminogen-deficient mice also exhibit the laminar band and DSD- 1-PG/phosphacan deposition, but mossy fiber outgrowth through the supragranular region is normal. These results demonstrate that tPA functions acutely, both through and independently of plasmin, to mediate mossy fiber reorganization.

Mice deficient for tenascin-R display alterations of the extracellular matrix and decreased axonal conduction velocities in the CNS.

Tenascin-R (TN-R), an extracellular matrix glycoprotein of the CNS, localizes to nodes of Ranvier and perineuronal nets and interacts in vitro with other extracellular matrix components and recognition molecules of the immunoglobulin superfamily. To characterize the functional roles of TN-R in vivo, we have generated mice deficient for TN-R by homologous recombination using embryonic stem cells. TN-R-deficient mice are viable and fertile. The anatomy of all major brain areas and the formation and structure of myelin appear normal. However, immunostaining for the chondroitin sulfate proteoglycan phosphacan, a high-affinity ligand for TN-R, is weak and diffuse in the mutant when compared with wild-type mice. Compound action potential recordings from optic nerves of mutant mice show a significant decrease in conduction velocity as compared with controls. However, at nodes of Ranvier there is no apparent change in expression and distribution of Na+ channels, which are thought to bind to TN-R via their beta2 subunit. The distribution of carbohydrate epitopes of perineuronal nets recognized by the lectin Wisteria floribunda or antibodies to the HNK-1 carbohydrate on somata and dendrites of cortical and hippocampal interneurons is abnormal. These observations indicate an essential role for TN-R in the formation of perineuronal nets and in normal conduction velocity of optic nerve.

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.

Glycosaminoglycans and glycoproteins associated with rat brain nuclei.

The concentration, composition and sulfate labeling of glycosaminoglycans and glycoproteins have been studied in purified nuclei isolated in bulk from rat brain. The concentration of total glycosaminoglycans is 0.142 mumol hexosamine/100 mg protein, comprising 57% chondroitin 4-sulfate, 7% chondroitin 6-sulfate, 29% hyaluronic acid and 7% heparan sulfate. Control experiments demonstrated that less than 5% of the sulfated glycosaminoglycans associated with nuclei could be accounted for by the nonspecific adsorption of soluble acidic proteoglycans to basic nuclear proteins. Glycoprotein carbohydrate is present at a level of 206 mug/100 mg protein, and has an average composition of 30% N-acetylglucosamine, 29% mannose, 19% N-acetylneuraminic acid, 15% galactose, 4% N-acetylgalactosamine, and 3% fucose. Labeling studies also indicated the presence of ester sulfate residues on the glycoprotein oligosaccharides.

Localization of epidermal hyaluronic acid using the hyaluronate binding region of cartilage proteoglycan as a specific probe.

Hyaluronate is actively synthesized by cultured epidermis and dermis, but no direct histological data have been available about its localization in normal human skin. A hyaluronate-specific biotinylated probe, prepared from the hyaluronate binding region of cartilage proteoglycan, was applied to human skin sections and visualized using the biotin-avidin-peroxidase system. The specificity of this staining was confirmed by hyaluronidase predigestion and by hyaluronate-derived oligosaccharides added to the staining solution. All dermis showed diffuse binding of the probe, but the highest staining intensity was observed in the epidermal intercellular spaces. The stainability extended from basal cells to the middle layers of the epidermis, whereas the granular layer and stratum corneum were completely negative. Also, the basal side of basal cells (basement membrane) did not bind the hyaluronate probe. The abundance of hyaluronate on surfaces and intercellular spaces of the spinous cells is suggested to have an important role in the physiology of human epidermis.

Hyaluronate accumulation in human epidermis treated with retinoic acid in skin organ culture.

Retinoic acid (RA) has been shown to retard the differentiation of epidermal keratinocytes by several morphologic and biochemical criteria. In this study, the epidermal content and localization of hyaluronate (HA), as well as its synthesis and disappearance in human skin organ culture, were characterized to test the idea that some of the RA influences on epidermal differentiation are associated with keratinocyte HA metabolism. RA stimulated the incorporation of 3H-glucosamine into HA by up to 60% at concentrations between 50 nM and 5 microM, while pulse-chase experiments revealed little change in its disappearance rate from epidermis. After 5 d in culture, the chemically quantified HA was more than doubled in the treated epidermis. The accumulation of HA was substantiated by light and electron microscopy with a specific probe prepared from the HA binding region of cartilage proteoglycan. The staining was particularly enhanced between the upper spinous cell layers, where the terminal differentiation into corneocytes normally takes place. A patchy, discontinuous staining was also seen in stratum granulosum and corneum layers, which are not stained at all in control cultures. The present study demonstrates that RA leads to an accumulation of HA in the superficial layers of epidermis by stimulating its synthesis in keratinocytes. This may account for the delay in terminal differentiation, and the weakened cohesion of the keratinocytes previously observed both in vivo and vitro.

Properties of nervous tissue proteoglycans relevant to studies on Alzheimer's disease.

Our comments concern certain properties of nervous tissue proteoglycans which were not emphasized in the review by Snow and Wight, with particular attention to the proposed relation of the amyloid beta protein precursor to a heparan sulfate proteoglycan core protein.

Fucosyl gangliosides of PC12 pheochromocytoma cells.

Three monosialogangliosides are highly labeled when PC12 pheochromocytoma cells are cultured in the presence of L-[3H]fucose, and two additional monosialogangliosides are labeled to a lesser extent. In contrast, neither of the two disialogangliosides of PC12 cells contains fucose residues. Removal of sialic acid and fucose by formic acid hydrolysis demonstrated the presence of 3 major 'core' structures in the monosialogangliosides, and a single asialo derivative of the disialogangliosides which has the same chromatographic mobility as one of the monosialoganglioside hydrolysis products. None of the major formic acid hydrolysis products of the PC12 cell gangliosides corresponds to asialo-GM1, supporting our previous conclusion that PC12 cells do not contain significant amounts of brain-type gangliosides.

Chondroitin sulfate proteoglycans in the developing central nervous system. I. cellular sites of synthesis of neurocan and phosphacan.

We have used in situ hybridization histochemistry to examine the cellular sites of synthesis of two major nervous tissue proteoglycans, neurocan and phosphacan, in embryonic and postnatal rat brain and spinal cord. Both proteoglycans were detected only in nervous tissue. Neurocan mRNA was evident in neurons, including cerebellar granule cells and Purkinje cells, and in neurons of the hippocampal formation and cerebellar nuclei. In contrast, phosphacan message was detected only in astroglia, such as the Golgi epithelial cells of the cerebellum. At embryonic day 13-16, phosphacan mRNA is largely confined to areas of active cell proliferation (e.g., the ventricular zone of the ganglionic eminence and septal area of the brain and the ependymal layer surrounding the central canal of the spinal cord) as well as being present in the roof plate. The distribution of neurocan message is more widespread, extending to the cortex, hippocampal formation, caudate putamen, and basal telencephalic neuroepithelium, and neurocan mRNA is present in both the ependymal and mantle layers of the spinal cord but not in the roof plate. The presence of neurocan mRNA in areas where the proteoglycan is not expressed suggests that the short open reading frame in the 5'-leader of neurocan may function as a cis-acting regulatory signal for the modulation of neurocan expression in the developing central nervous system.

Chondroitin sulfate proteoglycans in the developing central nervous system. II. Immunocytochemical localization of neurocan and phosphacan.

Using immunocytochemistry, we have compared the distribution of neurocan and phosphacan in the developing central nervous system. At embryonic day 13 (E13), phosphacan surrounds the radially oriented neuroepithelial cells of the telencephalon, whereas neurocan staining of brain parenchyma is very weak. By E16-19, strong staining of both neurocan and phosphacan is seen in the marginal zone and subplate of the neocortex, and phosphacan is present in the ventricular zone and also has a diffuse distribution in other brain areas. Phosphacan is also widely distributed in embryonic spinal cord, where it is strongly expressed throughout the gray and white matter, in the dorsal and ventral nerve roots, and in the roof plate at E13, when neurocan immunoreactivity is seen only in the mesenchyme of the future spinal canal. Neurocan first begins to appear in the spinal cord at E16-19, in the region of ventral motor neurons. In early postnatal and adult cerebellum, neurocan immunoreactivity is seen in the prospective white matter and in the granule cell, Purkinje cell, and molecular layers, whereas phosphacan immunoreactivity is associated with Bergmann glial fibers in the molecular layer and their cell bodies (the Golgi epithelial cells) below the Purkinje cells. These immunocytochemical results demonstrate that the expression of neurocan and phosphacan follow different developmental time courses not only in postnatal brain (as previously demonstrated by radioimmunoassay) but also in the embryonic central nervous system. The specific localization and different temporal expression patterns of these two proteoglycans are consistent with other evidence indicating that they have overlapping or complementary roles in axon guidance, cell interactions, and neurite outgrowth during nervous tissue histogenesis.