Fumiko Matsui

Developmentally regulated expression of a brain specific species of chondroitin sulfate proteoglycan, neurocan, identified with a monoclonal antibody 1G2 in the rat cerebrum

The mammalian brain contains many species of proteoglycan. To identify each proteoglycan species, we have raised monoclonal antibodies against soluble chondroitin sulfate proteoglycans purified from 10-day-old rat brains. One monoclonal antibody, named monoclonal antibody 1G2, recognized two proteoglycan species with 220,000 and 150,000 mol. wt core glycoproteins (chondroitin sulfate proteoglycan-220 and chondroitin sulfate proteoglycan-150). Partial amino acid sequences of N-termini of their core proteins coincided with those of neurocan, a brain-unique chondroitin sulfate proteoglycan species, whose complete coding sequence was recently reported [Rauch et al.(1992) J. biol. Chem.269, 19,536–19,547]. Western blots revealed that chondroitin sulfate proteoglycan-220 became detectable in the rat cerebrum on embryonic day 14, and that it disappeared from the brain around postnatal day 30. In contrast, a fairly large amount of chondroitin sulfate proteoglycan-150 remained in the mature brain. Immunohisto-chemical studies revealed that 1G2 antigen was first localized in the preplate zone, then both in the marginal zone and in the subplate of the rat cerebrum on embryonic day 16, prior to arrival of the first thalamic afferents at the cortex. On embryonic day 20, immunolabeling with monoclonal antibody 1G2 began to spread from the subplate into the developing cortical plate. On postnatal day 10, the neuropil of the cerebrum, except for the barrel field, was diffusely stained with the antibody, intensely in the hippocampus and superficial layers (I–III) of the cerebral cortex and weakly elsewhere. The barrel hollows were stained very weakly compared with the barrel walls at this stage. The immunoreactivity in the hippocampus and superficial cortical layers was weakened in the mature brain, so that no particular staining pattern, but weak and diffuse staining was observed in the adult rat cerebrum. The 1G2 antigen was immunohistochemically associated largely with glial fibrillary acidic protein-positive cells in primary cultures of the neonatal rat cerebrum. Both chondroitin sulfate proteoglycan-220 and chondroitin sulfate proteoglycan-150 were detected in the conditioned media not only of highly enriched cultures of fetal rat cortical neurons but also of pure cultures of mature astrocytes; more (12- to 20-fold) in the astrocyte conditioned media. Astrocytes, in addition to neurons, may be a cellular source of neurocan in brain at least under certain physiological conditions. The spaciotemporal expression pattern of 1G2 epitope-bearing proteoglycan, or neurocan, suggests that this proteoglycan species plays some roles at least in forming the elongation pathway for early cortical afferent fibers as well as the functional barrel structure in the somatosensory cortex.

Identification and Functions of Chondroitin Sulfate in the Milieu of Neural Stem Cells

The behavior of cells is generally considered to be regulated by environmental factors, but the molecules in the milieu of neural stem cells have been little studied. We found by immunohistochemistry that chondroitin sulfate (CS) existed in the surroundings of nestin-positive cells or neural stem/progenitor cells in the rat ventricular zone of the telencephalon at embryonic day 14. Brain-specific chondroitin sulfate proteoglycans (CSPGs), including neurocan, phosphacan/receptor-type protein-tyrosine phosphatase β, and neuroglycan C, were detected in the ventricular zone. Neurospheres formed by cells from the fetal telencephalon also expressed these CSPGs and NG2 proteoglycan. To examine the structural features and functions of CS polysaccharides in the milieu of neural stem cells, we isolated and purified CS from embryonic day 14 telencephalons. The CS preparation consisted of two fractions differing in size and extent of sulfation: small CS polysaccharides with low sulfation and large CS polysaccharides with high sulfation. Interestingly, both CS polysaccharides and commercial preparations of dermatan sulfate CS-B and an E-type of highly sulfated CS promoted the fibroblast growth factor-2-mediated proliferation of neural stem/progenitor cells. None of these CS preparations promoted the epidermal growth factor-mediated neural stem cell proliferation. These results suggest that these CSPGs are involved in the proliferation of neural stem cells as a group of cell microenvironmental factors.

A chondroitin sulfate proteoglycan that is developmentally regulated in the cerebellar mossy fiber system

It is known that the mammalian brain contains many kinds of proteoglycans, but almost all of them remain to be characterized. In this study, we prepared a monoclonal antibody against a phosphate-buffered saline-soluble brain proteoglycan (MAb 6B4). MAb 6B4 recognized a 600- to 1000-kDa chondroitin sulfate proteoglycan with a 250-kDa core protein (6B4 proteoglycan). The core protein of 6B4 proteoglycan carried the HNK-1 epitope. Immunohistochemical analysis of the adult rat brain indicated that this proteoglycan was expressed on the cell surfaces of a subset of neurons. In the hindbrain, 6B4 proteoglycan was highly expressed on the cerebellar Purkinje cells and Golgi cells, and at particular nuclei including the pontine nuclei and lateral reticular nucleus. Almost all of these nuclei were connected to the cerebellum through the mossy fiber system. A developmental study indicated that the expression of this proteoglycan changed dramatically during the formation of the cerebellar mossy fiber system. The mossy fibers from the pontine nuclei expressed 6B4 proteoglycan transiently from Embryonic Day 20 (E20) to Postnatal Day 30 (P30), during which time the axonal outgrowth and glomerular synapse formation occurred. The Purkinje cells, glomeruli, and Golgi cells began to be stained with MAb 6B4 from P10, P16, and P20, respectively. These expression stages correspond with the onset of their synapse formation. These results suggest that 6B4 proteoglycan is closely involved in the development of the cerebellar mossy fiber system.

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.

Brain development and multiple molecular species of proteoglycan

The occurrence of multiple proteoglycan species is a characteristic of the brain. The structural features of individually characterized proteoglycans in the brain are first introduced in brief, then some examples are shown that suggest a relationship between multiple proteoglycans and the many distinct cell types and neural circuits in the brain. Typical experiments demonstrated the neuronal-activity-dependent expression of neural proteoglycans during the critical developmental period of some functional systems such as the visual and vibrissal barrel systems. In addition, the binding properties of neural proteoglycans to other cell surface molecules are discussed in conjunction with their involvement in cell-cell and cell-substratum interactions. This review also covers other potential functions of proteoglycans not only in the development and maintenance of the brain but also in the pathogenesis of Alzheimers disease. Proteoglycans are really coming of age in neuroscience.

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.

Developmental change in the glycosaminoglycan composition of the rat brain.

Abstract: Glycosaminoglycans (GAGs) were isolated from the brains of pre‐ and postnatal rats. The GAG content of the brain, based on the amount of DNA, was constant during the period from day 13 to day 15 of gestation. After day 15, the GAG content began to increase and reached a plateau by 10 days after birth. Hyaluronate (HA) was the main GAG (> 60% of the total) in the fetal rat brain, and the relative amount of HA decreased after birth. Conversely, the relative amount of chondroitin sulfate increased with development and reached the adult level by 20 days after birth. Heparan sulfate (HS) was the major sulfated GAG in the fetal rat brain at early developmental stages, but HS accounted for approximately 10% of the total GAG in the postnatal brains. In addition to these GAGs, a polysialosyl glycoconjugate was isolated from rapidly growing brains of the rat. These three GAGs could be isolated either from the cerebellum, cerebrum, or brainstem of the newborn rat. A closely similar age‐related change in the GAG composition was observed in each of these different regions of the brain. The developmental change could be implicated in morphogenesis or maturation of the brain.

Neuroglycan C, a Novel Membrane-spanning Chondroitin Sulfate Proteoglycan That Is Restricted to the Brain

Monoclonal antibodies were raised to membrane-bound proteoglycans derived from rat brain, and four monoclonal antibodies that recognized a 150-kDa chondroitin sulfate proteoglycan with a core glycoprotein of 120 kDa were obtained. Immunohistological study revealed that the proteoglycan was associated with developing neurons. We screened rat brain cDNA libraries using the four monoclonal antibodies and isolated overlapping cDNA clones that encoded the entire core protein of 514 amino acids plus a 30-residue signal peptide. The deduced amino acid sequence suggested an integral membrane protein divided into five structurally different domains: an N-terminal domain to which chondroitin sulfate chains might be attached, a basic amino acid cluster consisting of seven arginine and two lysine residues, a cysteine-containing domain, a membrane-spanning segment, and a C-terminal cytoplasmic domain of 95 amino acids. On Northern blots, the cDNA hybridized with a single mRNA of 3.1 kilobases that was detectable in brains of neonatal and adult rats but not in kidney, liver, lung, and muscle of either. The sequence of the proteoglycan did not exhibit significant homology to any other known protein, indicating that the proteoglycan, designated neuroglycan C, is a novel integral membrane proteoglycan.

Immunological identification of two proteoglycan fragments derived from neurocan, a brain-specific chondroitin sulfate proteoglycan

Neurocan is a brain-unique chondroitin sulfate proteoglycan (CSPG) whose expression and proteolytic cleavage are developmentally regulated. One of the proteolytic products (C-terminal half) is known to be a CSPG with a 150 kDa core glycoprotein (CSPG-150). To identify the N-terminal half of neurocan, we raised an anti-neurocan polyclonal antibody (PAb 291) using a synthetic peptide whose amino acid sequence matched a part of the N-terminal half of neurocan. Western blots showed that PAb 291 recognized two CSPGs, one with a 220 kDa core glycoprotein (CSPG-220, namely neurocan) and one with a 130 kDa core glycoprotein (CSPG-130) isolated from young rat brains. CSPG-130 was co-purified along with CSPG-220 by PAb 291-immunoaffinity column chromatography. The amino acid sequence of the N-terminus of the immunopurified CSPG-130 was exactly the same as the N-terminal sequence of CSPG-220. These results suggest that not only the C-terminal half (CSPG-150) but also the N-terminal half (CSPG-130) of CSPG-220 exists in a CSPG form in rat brain. Using PAb 291 and monoclonal antibody 1G2 (MAb 1G2) which recognizes CSPG-150 in addition to CSPG-220, we found that the contents of CSPG-130 and CSPG-150 in the rat brain reached maximum levels around the time of birth. Both CSPG-130 and 150 were observed, while CSPG-220 was hardly detectable in extracts from the adult rat brain. Immunohistochemical investigation showed that the PAb 291 antigen had a similar distribution pattern to the MAb 1G2 antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Proteoglycans and injury of the central nervous system

ABSTRACT  Proteoglycan is a family of glycoproteins which carry covalently‐linked glycosaminoglycan chains, such as chondroitin sulfate and heparan sulfate. Proteoglycans are believed to play important roles in morphogenesis and maintenance of various tissues including the central nervous system (CNS) through interactions with cell adhesion molecules and growth factors. In the CNS, a significant amount of evidence has been accumulated to show that proteoglycans function as modulators in various cellular events not only in the development, but also in the pathogenesis of neuronal diseases and lesions. When the CNS is injured, several chondroitin sulfate proteoglycans (CSPG) are up‐regulated in glial scars formed around the lesion site. The glial scar also contains some molecules inhibitory to axonal growth, such as myelin‐associated glycoprotein, Nogo, and Semaphorin. In vitro studies revealed that CSPG largely exert a repulsive effect on axonal regeneration, and a signal from CSPG modulates the actin cytoskeleton of outgrowing neurites through the Rho/ROCK pathway. These findings suggest that CSPG are responsible for unsuccessful axonal regeneration in glial scars. Various attempts to overcome the inhibitory effect of CSPG have been pursued in vivo. Digestion of chondroitin sulfate chains by chondroitinase ABC, suppression of CSPG core protein synthesis by decorin, suppression of glycosaminoglycan chain synthesis by a DNA enzyme, and inhibition of the Rho/ROCK pathway with specific inhibitors were all successful for increasing axonal regeneration. For a clinical application, the most effective combination of these treatments needs to be examined in the future.