Monoclonal antibody Cat-315 detects a glycoform of receptor protein tyrosine phosphatase beta/phosphacan early in CNS development that localizes to extrasynaptic sites prior to synapse formation.

Perineuronal nets (PNs) are lattice-like condensations of the extracellular matrix (ECM) that envelop synapses and decorate the surface of subsets of neurons in the CNS. Previous work has suggested that, despite the fact that PNs themselves are not visualized until later in development, some PN component molecules are expressed in the rodent CNS even before synaptogenesis. In the adult mammalian brain, monoclonal antibody Cat-315 recognizes a glycoform of aggrecan, a major component of PNs. In primary cortical cultures, a Cat-315-reactive chondroitin sulfate proteoglycan (CSPG) is also expressed on neuronal surfaces and is secreted into culture media as early as 24 h after plating. In this study, we show that in primary cortical cultures, the Cat-315 CSPG detected in early neural development is expressed in extrasynaptic sites prior to synapse formation. This suggests that ECM components in the CNS, as in the neuromuscular junction (NMJ), may prepattern neuronal surfaces prior to innervation. We further show that while the Cat-315-reactive carbohydrate decorates aggrecan in the adult, it decorates a different CSPG in the developing CNS. Using receptor protein tyrosine phosphatase beta (RPTPbeta/protein tyrosine phosphatase zeta) knock-out mice and immunoprecipitation techniques, we demonstrate here that in the developing rodent brain Cat-315 recognizes RPTPbeta isoforms. Our further examination of the Cat-315 epitope suggests that it is an O-mannose linked epitope in the HNK-1 family. The presence of the Cat-315 reactive carbohydrate on different PN components--RPTPbeta and aggrecan--at different stages of synapse development suggests a potential role for this neuron-specific carbohydrate motif in synaptogenesis.

Brain enriched hyaluronan binding (BEHAB)/brevican increases aggressiveness of CNS-1 gliomas in Lewis rats.

Gliomas are the most common primary intracranial tumors. One extracellular matrix component that has been implicated in glial tumor biology is brain enriched hyaluronan binding (BEHAB)/brevican. In this study, the CNS-1 rat glioma cell line was transfected with a vector containing either a full-length BEHAB/brevican cDNA, a 5' insert encoding the NH(2)-terminal BEHAB/brevican cleavage product, or a 3' insert encoding the COOH-terminal cleavage product. As a control, CNS-1 cells were transfected with green fluorescent protein. Rats with intracranial grafts of BEHAB/brevican-transfected CNS-1 cells displayed significantly shorter survival times than did rats with CNS-green fluorescent protein intracranial grafts (P < 0.001). Histological examination showed that the BEHAB/brevican-transfected tumors were just as, if not more, aggressive than control tumors, even though the BEHAB/brevican tumors had been growing for only approximately two-thirds the time as long as control tumors. These data suggest that up-regulation and proteolytic cleavage of BEHAB/brevican increase significantly the aggressiveness of glial tumors. It will be important to investigate the effect of inhibiting cleavage of BEHAB/brevican in these cells and to determine the therapeutic potential of inhibiting BEHAB/brevican cleavage in gliomas.

Glial tumor invasion: a role for the upregulation and cleavage of BEHAB/brevican.

Glial tumors, gliomas, are the most common primary intracranial tumors. Their distinct ability to invade the normal surrounding tissue makes them difficult to control and nearly impossible to completely remove surgically, and it accounts for the extraordinarily high lethality associated with gliomas. The ability of these transformed glial cells to invade the normal surrounding tissue is relatively unique in the adult CNS, which under most circumstances, is inhibitory to cell movement. The extracellular matrix (ECM) can modulate, in part, the permissiveness of a tissue to cell movement. Accordingly, the ability of gliomas to modify the ECM of the CNS may mediate the invasiveness of these cells. One ECM molecule that shows dramatic upregulation in gliomas is BEHAB (brain enriched hyaluronan binding)/brevican, a brain-specific chondroitin sulfate proteoglycan. BEHAB/brevican expression is also upregulated during periods of increased glial cell motility in development and following brain injury. Experimental evidence suggests that in glioma, in addition to upregulation of BEHAB/brevican, proteolytic processing of the full-length protein also may contribute to invasion. Here, the authors present a review of the literature on glial tumor invasion by modulation of the ECM and propose a two-step model for BEHAB/brevican's role in this process.

cDNA cloning, chromosomal localization, and expression analysis of human BEHAB/brevican, a brain specific proteoglycan regulated during cortical development and in glioma.

BEHAB (Brain Enriched HyAluronan Binding)/brevican, a brain-specific member of the lectican family of chondroitin sulfate proteoglycans (CSPGs), may play a role in both brain development and human glioma. BEHAB/brevican has been cloned from bovine, mouse and rat. Two isoforms have been reported: a full-length isoform that is secreted into the extracellular matrix (ECM) and a shorter isoform with a sequence that predicts a glycophosphatidylinositol (GPI) anchor. Here, we report the characterization of BEHAB/brevican isoforms in human brain. First, BEHAB/brevican maps to human chromosome 1q31. Second, we report the sequence of both isoforms of human BEHAB/brevican. The deduced protein sequence of full-length, secreted human BEHAB/brevican is 89.7, 83.3 and 83.2% identical to bovine, mouse and rat homologues, respectively. Third, by RNase protection analysis (RPA) we show the developmental regulation of BEHAB/brevican isoforms in normal human cortex. The secreted isoform is highly expressed from birth through 8years of age and is downregulated by 20years of age to low levels that are maintained in the normal adult cortex. The GPI isoform is expressed at uniformly low levels throughout development. Fourth, we confirm and extend previous studies from our laboratory, here demonstrating the upregulation of BEHAB/brevican mRNA in human glioma quantitatively. RPA analysis shows that both isoforms are upregulated in glioma, showing an approximately sevenfold increase in expression over normal levels. In contrast to the developmental regulation of BEHAB/brevican, where only the secreted isoform is regulated, both isoforms are increased in parallel in human glioma. The distinct patterns of regulation of expression of the two isoforms suggest distinct mechanisms of regulation of BEHAB/brevican during development and in glioma.

Brain-enriched hyaluronan binding (BEHAB)/brevican cleavage in a glioma cell line is mediated by a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family member.

Brain-enriched hyaluronan binding (BEHAB)/brevican is a brain-specific extracellular matrix protein containing a cleavage site between Glu(395)-Ser(396), which bears remarkable homology to the "aggrecanase" site in the cartilage proteoglycan aggrecan. Expression of BEHAB/brevican is dramatically increased in human gliomas, notoriously invasive tumors. Recently, we showed that the rat 9L gliosarcoma cell line, which does not express BEHAB/brevican and forms non-invasive tumors when grown as intracranial grafts, can form invasive tumors when transfected with a 5' cDNA fragment of BEHAB/brevican, but not when transfected with the full-length cDNA. In marked contrast, the highly invasive CNS-1 glioma cell line expresses and cleaves BEHAB/brevican protein when grown as an intracranial graft. These results suggest that both synthesis and cleavage of BEHAB/brevican protein may play a role in the invasiveness of gliomas. We report here, using an antibody developed to the neoepitope created by BEHAB/brevican cleavage at the Glu(395)-Ser(396) site, that the CNS-1 cells are able to cleave the protein in vitro. We characterized the CNS-1-derived cleavage activity by assaying its ability to cleave BEHAB/brevican proteoglycan, and determined that the enzyme is a constitutively expressed, secreted activity. Using a variety of protease inhibitors, reverse transcriptase-polymerase chain reaction, and specific antibodies, we determined that this activity is likely to be a member of the ADAMTS family of metalloproteinases, specifically ADAMTS4. These results suggest a novel function for ADAMTS family members in BEHAB/brevican cleavage and glioma and indicate that inhibition of ADAMTS in glioma may provide a novel therapeutic strategy.

BEHAB/brevican: an extracellular matrix component associated with invasive glioma.

The extracellular matrix (ECM) has a prominent role in many physiological processes, including organ development, wound healing, and neoplastic growth and invasion. In each of these processes, changes in the composition of the matrix can lead to increased cell movement. In this review, we discuss the role of ECM components in glioma invasion, with special emphasis on the brain-specific proteoglycan, Brain-Enriched Hyaluronan Binding (BEHAB)/brevican.

A family of proteins implicated in axon guidance and outgrowth.

Rapid progress in the identification and characterization of axon guidance molecules and their receptors has left the field poised to explore the intracellular mechanisms by which signals are transduced into growth cone responses. The TUC (TOAD/Ulip/CRMP) family of proteins has emerged as a strong candidate for a role in growth cone signaling. The TUC family members reach their highest expression levels in all neurons during their peak periods of axonal growth and are strongly down-regulated afterward. When axonal regrowth in the adult is triggered by axotomy, TUC-4 is reexpressed during the period of regrowth. Mutations in unc-33, a homologous nematode gene, lead to severe axon guidance errors in all neurons. Furthermore, the TUC family is required for the growth cone-collapsing activity of collapsin-1. An important role for the TUC family is also suggested by its high degree of interspecies amino acid sequence identity, with the rat TUC-2 protein showing 98% identity with its chick ortholog and 89% identity with its Xenopus ortholog. Information gained from the study of the TUC family will be of key importance in understanding how growth cones find their targets.

Intracranial injury acutely induces the expression of the secreted isoform of the CNS-specific hyaluronan-binding protein BEHAB/brevican.

Hyaluronan (HA) plays an important role in tissue reorganization in response to injury. The mechanisms by which HA participates in these processes are likely to include HA-binding proteins. Previously, we reported the cloning and initial characterization of a central nervous system (CNS)-specific HA-binding protein, BEHAB (brain enriched hyaluronan binding), which was independently cloned in another laboratory and named brevican. BEHAB/brevican mRNA is expressed in the ventricular zone coincident with the initial proliferation and migration of glial cells and in surgical samples of human glioma, where glial-derived cells proliferate and migrate. To determine whether BEHAB/brevican is also expressed during the cellular proliferation and migration associated with CNS injury, we have examined BEHAB/brevican expression during reactive gliosis. BEHAB/brevican occurs as secreted and cell-surface, glycosylphosphatidylinositol (GPI)-anchored, isoforms. The secreted, but not the GPI-anchored, isoform is up-regulated in response to a stab wound to the adult rat brain. The temporal regulation and spatial distribution of BEHAB/brevican expression parallel the gliotic response and the expression of the intermediate filament protein nestin. The up-regulation of BEHAB/brevican in response to CNS injury suggests a role for this extracellular matrix molecule in reactive gliosis. Glial process extension, a central element in the glial response to injury, may require the reexpression of both cytoskeletal and matrix elements that are normally expressed during the glial motility seen in the immature brain.

BEHAB/brevican: a brain-specific lectican implicated in gliomas and glial cell motility.

Several recent findings have advanced our understanding of the composition and function of the brain extracellular matrix (ECM). BEHAB/brevican, a recently identified CNS-specific proteoglycan, is a component of the brain ECM and is upregulated during glial cell motility. It is expressed at high levels during development, in response to injury, and in primary brain tumors. Cleavage of the BEHAB/brevican protein may increase invasion of tumor cells.

Expression of a cleaved brain-specific extracellular matrix protein mediates glioma cell invasion In vivo.

Malignant gliomas (primary brain tumors) aggressively invade the surrounding normal brain. This invasive ability is not demonstrated by brain metastases of nonglial cancers. The brain-specific, brain-enriched hyaluronan binding (BEHAB)/brevican gene, which encodes an extracellular hyaluronan-binding protein, is consistently expressed by human glioma and is not expressed by tumors of nonglial origin (Jaworski et al., 1996). BEHAB/brevican can be cleaved into an N-terminal fragment that contains a hyaluronan-binding domain (HABD) and a C-terminal fragment (Yamada et al., 1995). Here, using antisera to peptides in the predicted N-terminal and C-terminal proteolytic fragments, we demonstrate that the BEHAB/brevican protein is cleaved in invasive human and rodent gliomas. A role for this protein in glioma cell invasion was tested by transfecting a noninvasive cell line with the BEHAB/brevican gene. The noninvasive 9L glioma cell was transfected with either full-length BEHAB/brevican or the HABD and tested for invasion in in vitro and in vivo invasion assays. Although both constructs increased invasion in vitro, only the HABD increased invasion by tumors growing in vivo. Experimental intracranial tumors from full-length transfectants showed no increase in invasion over control tumors, whereas tumors from HABD transfectants showed a marked potentiation of tumor invasion, producing new tumor foci at sites distant from the main tumor mass. This work demonstrates a role for a brain-specific extracellular matrix protein in glioma invasion, opening new therapeutic avenues for a uniformly fatal disease.

Neurons produce a neuronal cell surface-associated chondroitin sulfate proteoglycan.

Monoclonal antibody Cat-315 recognizes a chondroitin sulfate proteoglycan (CSPG) expressed on the surface of subsets of neurons in many areas of the mammalian CNS (). The cell type-specific expression exhibited by the Cat-315 CSPG and other perineuronal net CSPGs imparts a distinct molecular surface identity to a neuron (Celio and Blumcke, 1994; Lander et al., 1997). The cell type(s) producing these surface-associated proteins and yielding this cellular diversity has remained in question. The expression of the Cat-315 CSPG in primary rat cortical cultures has permitted an examination of the cellular source of the Cat-315 antigen, as well as a determination of its spatial relationship to the neuronal surface. Live-cell labeling of primary neuronal cultures demonstrates that the Cat-315 CSPG is on the extracellular surface of neurons. Furthermore, extraction experiments demonstrate that the Cat-315 CSPG lacks a transmembrane domain and that the entire molecule is extracellular and, therefore, can be considered a constituent of brain extracellular matrix. Several lines of evidence indicate that neurons with cell surface staining produce the Cat-315 CSPG. First, neurons with cell surface staining also show intracellular Cat-315 immunoreactivity. Second, beta-xyloside or monensin, reagents that inhibit the synthesis and transport of CSPGs, increase intracellular Cat-315 immunoreactivity within neurons that express cell surface Cat-315 immunoreactivity. Third, double labeling with Cat-315 and a polyclonal antibody for the Golgi complex demonstrates a precise colocalization of the intracellular Cat-315 immunoreactivity with the Golgi. Together, these observations demonstrate that neurons contribute to the extracellular matrix of brain and that the Cat-315 CSPG is produced by the neurons that carry Cat-315 cell surface immunoreactivity.

Glial cells assemble hyaluronan-based pericellular matrices in vitro.

The extracellular matrix (ECM) of the brain contains hyaluronan and proteoglycans, as does the ECM of cartilage. Aggrecan, the major proteoglycan of cartilage, forms large aggregates with hyaluronan, which then associate with the chondrocyte cell surface through an interaction with surface hyaluronan binding proteins. In culture, chondrocytes elaborate hyaluronan-proteoglycan aggregates, which form large hydrated pericellular matrices (PCMs) that can be visualized by a particle exclusion assay (Knudson and Toole: Dev Biol 112:308, 1985). It has recently been demonstrated that embryonic glial cells can also elaborate PCMs in culture (Deyst and Toole: Dev Brain Res 28:351, 1995). We demonstrate here that different classes of glial cells elaborate different types of endogenous PCMs in culture. Less differentiated glial cells, as evidenced by their immunoreactivity for nestin, elaborate larger endogenously produced PCMs than differentiated astrocytes, as defined by immunoreactivity for GFAP. This in vitro result may be a reflection of the larger volume of extracellular space present in the embryonic than in the mature brain. We show further that glial cells can incorporate cartilage aggrecan into their PCMs, and that both endogenous and aggrecan-supplemented glial PCMs are dependent on hyaluronan. In contrast, primary neurons from newborn (P0) and P1 rat cortex neither express endogenous matrices nor can assemble exogenous hyaluronan/aggrecan aggregates into PCMs. These results suggest that immature neurons may not have the ability to assemble hyaluronan-based PCMs, and they raise the possibility that neural proteoglycans associate with neuronal surfaces through a mechanism that may not directly involve hyaluronan.

Development of Cat-301 immunoreactivity in auditory brainstem nuclei of the gerbil.

The developing brainstem auditory system has been studied in detail by using anatomical and physiological techniques. However, it is not known whether immature auditory neurons exhibit different molecular characteristics than those of physiologically mature neurons. To address this issue, we examined the distribution of Cat-301 immunoreactivity in the developing auditory brainstem of gerbils. Cat-301 is a monoclonal antibody that recognizes a 680-kD chondroitin sulfate proteoglycan similar to aggrecan, a high-molecular-weight chondroitin sulfate proteoglycan found in cartilage. In the central nervous system, Cat-301 immunoreactivity is localized to the extrasynaptic surface of neurons. It has been hypothesized by Hockfield and co-workers (Hockfield et al. [1990a]Cold Spring Harbor Symp. Quart. Biol. 55:504-514) that the Cat-301 proteoglycan is a molecular marker indicating that a neuron has acquired mature neuronal properties. In the current study, Cat-301 staining is first seen at 7 days after birth in the anterior ventral cochlear nucleus (AVCN), the posterior VCN (PVCN), and the medial nucleus of the trapezoid body (MNTB) shortly before the onset of sound-evoked activity. By 21 days after birth, neurons in the AVCN, the PVCN, and the lateral and medial superior olive have attained adult-like distributions of Cat-301 staining concomitant with the physiological maturation of these neurons. Neurons in MNTB attain adult-like distributions of Cat-301 immunoreactivity at 1 year. The maturation of Cat-301 immunoreactivity parallels the physiological maturation of gerbil auditory neurons, and the Cat-301 proteoglycan may play a role in the formation and/or stabilization of auditory synapses.

A family of activity-dependent neuronal cell-surface chondroitin sulfate proteoglycans in cat visual cortex.

Monoclonal antibody Cat-301 recognizes a chondroitin sulfate proteoglycan (CSPG) expressed on the extracellular surface of cell bodies and proximal dendrites of specific subsets of neurons in many areas of the mammalian CNS, including the cat visual cortex. The Cat-301 CSPG is first detected at the close of the critical period in development, a period during which the pattern of neuronal activity determines the mature synaptic circuitry and neuronal phenotype. In the cat visual cortex, dark-rearing from birth prolongs the duration of the critical period and attenuates the expression of the Cat-301 antigen, implicating the Cat-301 CSPG in the cellular mechanisms that terminate the period of synaptic plasticity. Because the Cat-301 antigen is expressed on only a limited subset of neurons, we have further examined the molecular heterogeneity among neuronal cell-surface CSPGs and have asked (1) whether other neuronal subsets carry distinct CSPGs and (2) whether the activity-dependent expression of the Cat-301 CSPG is a property generalizable to related cell-surface CSPGs. Here, we report two new monoclonal antibodies, Cat-315 and Cat-316, which together with Cat-301 define a family of at least seven related yet distinct CSPGs. These three antibodies define nonidentical subsets of neurons in the cat visual cortex. The expression of normal levels of these CSPGs is reduced by dark-rearing. Together, these data show that the family of cell-surface CSPGs is molecularly diverse, that different sets of neurons express distinct complements of cell-surface antigens, and that the regulation of CSPG expression by activity may be a general feature of neuronal cell-surface CSPGs.

Phospholipase C-beta1 is present in the botrysome, an intermediate compartment-like organelle, and Is regulated by visual experience in cat visual cortex.

Monoclonal antibody Cat-307 identifies a 165 kDa neuronal protein expressed in the cat visual cortex during the period of sensitivity to alterations in visual experience (). Dark-rearing, which prolongs the sensitive period, also prolongs the expression of the Cat-307 protein. The Cat-307 protein localizes to an organelle, here called the botrysome (from the Greek botrys, cluster of grapes), that is located between the endoplasmic reticulum (ER) and Golgi apparatus. The botrysome is composed of small ring-shaped profiles with electron-dense coats. The size and morphology of the rings and their coats are similar to those described for ER to Golgi transport vesicles. Biochemically, the Cat-307 protein cofractionates with microsomes and partitions with subunits of the coatomer proteins that coat ER-to-Golgi transport vesicles. Partial amino acid sequencing reveals that the Cat-307 protein is phospholipase C-beta1, the G-protein-dependent phosphodiesterase that hydrolyses phosphatidylinositol 4,5 biphosphate into inositol 1,4,5 triphosphate and diacylglycerol after the stimulation of a variety of neurotransmitter receptors at the cell surface. These results suggest a role for phospholipase C-beta1 and the botrysome in developmental plasticity and provide a possible link between receptor activation at the cell surface and protein transport during neuronal development.

Cues intrinsic to the spinal cord determine the pattern and timing of primary afferent growth.

We have used organotypic cultures of embryonic rat spinal cord and dorsal root ganglia (DRG) to study the development of central projections of primary sensory afferent axons that express calcitonin gene-related peptide (CGRP). In vivo, small- and medium-diameter CGRP-positive primary afferents terminate in laminae I, II, and V of the spinal cord and do not enter the ventral horn. A similar pattern of CGRP-positive axonal projections was observed in spinal cord slices of Day 16 embryos (E16) maintained in culture for 6 days. Both intact and dissociated DRG neurons showed the same pattern of central arborization, indicating that complex intercellular interactions between DRG neurons are not required for laminar specific targeting. Furthermore, targeting to the dorsal horn and avoidance of the ventral horn was observed in isolated dorsal and ventral hemicords, suggesting that separate mechanisms mediate the avoidance of CGRP-positive axons from the ventral horn and the elaboration of the afferent arbors within the dorsal horn. CGRP-positive afferents can grow into the dorsal horn only during a brief time window. Cultures of age-matched (isochronic) DRG and spinal cord from E14, E16, and E18 animals showed the characteristic pattern of CGRP-positive axon arborization, while cultures from E20 and neonatal animals did not. Heterochronic cultures indicate that it is the age of the spinal cord, and not the age of the DRG, that determines the ability of the CGRP-positive afferents to arborize within the dorsal horn. Together these results demonstrate that cues intrinsic to the spinal cord can direct sensory projections to appropriate locations in the spinal cord.

Identification and characterization of novel developmentally regulated proteins in rat spinal cord.

We previously used 2-dimensional (2-D) gel electrophoresis to identify novel proteins that may be involved in the genesis of the mammalian nervous system [1]. Several novel proteins that were up- or down-regulated coincident with neurogenesis and neuronal migration in rat neocortex were identified. To further investigate the expression of some of these developmentally regulated proteins during a comparable period in spinal cord development, 2-D electrophoresis is used to study their regulation in the crude membrane and soluble fractions of spinal cord at embryonic day 12 (E12) and embryonic day 21 (E21). This analysis indicates that 7 of the proteins that exhibited large changes in their synthesis in cerebral cortex between embryonic day 14 (E14) and embryonic day 21 (E21) demonstrate similar up- or down-regulation during spinal cord neurogenesis. However, two proteins are restricted in their expression or developmental regulation. One of these, 667-800, appears cortex-specific, while the up-regulation of protein SC.1 appears to be spinal cord specific. Several of these proteins also appear to be enriched in both the cortex and spinal cord relative to non-neural tissues (117, 162, 182, 310 [TOAD-64], 667-800) and may be neural specific. To further characterize its expression, one of these neural-specific, up-regulated proteins, TOAD-64 (protein 310) [2-4], is studied throughout embryonic and postnatal spinal cord development using peptide-specific polyclonal antibodies. As suggested by the 2-D gel analysis and the previously reported expression pattern in cerebral cortex [3], TOAD-64 is transiently expressed in postmitotic spinal cord neurons early in their development and sharply down-regulated after the second postnatal week. In the adult spinal cord, TOAD-64 expression is remarkably restricted to a subset of primary afferents to the spinal cord. This expression pattern, coupled with its recently discovered homology to two proteins implicated in axon pathfinding in the chick and nematode [5,3], suggests that TOAD-64 may have a fundamental role in axon pathfinding.