Proteoglycans in the nervous system

Proteoglycans are ubiquitous ceil-surface and secreted glycoproteins that are involved in diverse cellular behaviors. The identities of several nervous system proteoglycans, including many of the major species in the mammalian brain, have recently come to light. In addition, recent studies have given new insights into the roles of proteoglycans in nervous system development and function. Current Opinion Neurobiology In this provocative study, an HSPG was isolated from mouse neuron epithelial cells at two stages of development, and tested for binding to FGF-1 and FGF~2. HSPG from embtyonic day 9 cells preferentialb bound FGF-2, whereas HSPG from day 11 cells preferentially bound FGF~l. Similar speciIicity was seen in the ability of the HSPG samples to potentiate the actions of FGF-1 and FGF-2. The developmental switch in binding coincides with a switch in expression of FGFs in the neuro-epithelium, from FGF-2 to FGF~l, over the same period. This study demonstrates that a ceil surface HSPG can directly mediate cell adhesion, and that a single HSPG can have different affinities for an ECM l&and, depending, presumably, on the structure of its GAG chains. This paper raises the provocative hypothesis that the disappearance of CS from the developing mammalian retina sets up a spatial gradient that guides ganglion cell axons toward the optic nerve head. Preti~ ous in uitro studies had suggested that CS repels retinal axons. In this study, evidence is presented that newly differentiating ganglion cells find themselves within a steep CS gradient oriented away from the optic disk. Evidence is also presented that exposure of developing retinas to a CS-degrading enzyme causes large perturbations in ganglion cell axon guidance, as well as causing ectopic differentiation of ganglion cells.


Introduction
Proteoglycans (PGs) are found on the surfaces of all adherent cells, within intracellular vesicles, and in virtually all extracellular matrices (ECMS). They are evolutionarily ancient molecules, and play functional roles in the biology of growth factors, extracellular proteolysis, cell adhesion, lipoprotein metabolism, and virus entry into cells, as well as structural roles in maintaining the physical and mechanical properties of ECMs [ 1,2*,3,4], Although many basic characteristics of PGs -their number, their structures, the exact nature of the functions they perform -are still slowly emerging, great progress has been made in recent years. With this recent burst of activity has come increasing recognition of the significance of PGs by neurobiologists, and increasing interest in the postulated roles PGs play in the nervous system. Some investigators have isolated monoclonal antibodies against nervous system molecules that have turned out to be PGs. Other investigators have become intrigued by the fact that many of the molecules that are thought to inlluence neuronal and glial cell behavior in vivo, especially during development, bind PGs. In the last few years, direct assaults on determining the structures of central nervous system (CNS) PGs have been undertaken by several groups. The purpose of this article is to review some of these recent results, and place them into the wider context of what PGs are, and how they are thought to function.

What are PCs?
A protein is called a PG if it contains a covalently attached glycosaminoglycan (GAG). GAGS are linear polysaccharides, typically 20-200 sugars in length, which are usually attached via a characteristic linkage region to serine residues. GAGS are built by the sequential addition of identical disaccharide units onto this linkage region. Only three types of disaccharide may be used, giving rise to three families of GAGS: the heparin/heparan family [ D-glucuronic acid p(1+4) D-Nacetyl glucosamine a( l-4)] *; the chondroitin/dermatan family [D~glucuronic acid [3(1+3) D-N-ace@ galactosamine p( l-+4)] n; and the keratan family [ D-galactose p(1+4) D-N-acetyl glucosamine 0(1+3)],.
The sugars of most GAGS are further chemically modified, typically in a sporadic fashion throughout the chain, by Osulfation, N deacetylation followed by N-sulfation, and/or epimerization (isomerization) of glucuronic acid to iduronic acid. Subsequently, GAGS are referred to as heparin, heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (DS) or keratan sulfate (KS). The heparin/HS distinction and the CS/DS distinction only reflect differences in level of modification (i.e. heparin is more highly modified than most HS species; DS contains much more iduronate than CS). As each disaccharide in a GAG chain may be modified to a different degree, the large scale structures of GAGS can be exceedingly complex (e.g. in HS, which can be modified in up to five ways, a hexasaccharide can theoretically have over 30,000 possible chemical structures).
Products of several gene families, including secreted and membrane-inserted polypeptides, act as the core proteins of major PGs (Table 1). Some bear as few as one GAG chain, whereas others have over a hundred. Although the signals that specify whether a serine residue will bear a GAG are partially understood [2*], it is not known what controls the type of GAG synthesized: examples exist of cores that always bear one type of GAG, cores that bear different GAGS at different sites, and cores that bear different GAGS depending on the cell type in which they are expressed.  Table 2). In the embryo, glypican is also strongly expressed in ventricular zones (regions undergoing neural precursor proliferation) throughout the neuraxis (Fig. 1).

Cerebroglycan
Cerebroglycan, previously called PG Ml3 [6], is an HSPG with a -58 kDa core protein, and was first detected in the embryonic and newborn -but not adult -rat brain. Like glypican, it is glycosylphosphatidylinositolanchored. In fact, glypican and cerebroglycan define a family of lipid-anchored HSPG cores, based on amino acid sequence similarity (CS Stipp, ED Litwack, AD Iander, unpublished data) (see Table 2). In situ hybridization studies indicate that cerebroglycan is transiently expressed by postmitotic neurons throughout the CNS (Fig.  1). Evidently, cerebroglycan mRNA appears in neurons shortly after terminal mitosis and disappears after neuronal migration and axon growth have been completed. Interestingly, cerebroglycan is not expressed outside the nervous system.

N-syndecan
N-syndecan (or syndecdn-3) is one of four members of the syndecan family of transmembrane core proteins (Table 1). These polypeptides have short (-34 amino acids) cytoplasmic domains that are highly conserved among all family members, and overall sizes varying from 20 kDa (syndecans-2 and -4) to 2 42 kDa (syndecan-3). Their extracellular domains are poorly conserved among the different family members, or even for the same syndecan in different mammalian species. N-syn decan was cloned by Carey et al. [9**], who identified it in rat Schwann cell membranes (see Table 2). High levels of Nsyndecan mRNA are also found in neonatal rat brain, as well as in many sites outside the nervous system. Expression of this molecule in rat brain peaks at birth, declining to undetectable levels thereafter. Early immunohistochemical studies suggest that this PG is associated with fiber tracts, but it is not yet known whether its source is neuronal or glidl.

Syndecan-2
Syndecan-2, also known as fibroglycan, another member of the syndecan family, has not yet been isolated from the brain, but its mRNA has been found there (see Table  2). Based on electrophoretic behavior, syndecan-2 may correspond to brain PG Ml4 [6]. . In many cases, information on CNS distribution has been based on the examination of only a few brain regions, and is therefore incomplete. bData on distribution of glypican and cerebroglycan are based on rn situ hybridization; most other data were obtained using antibodies.

NC2
NG2 is a transmembrane CSPG with a 300 kDa core protein [ 101. In the brain it is associated with a population of glial precursor cells, the O-2A progenitors (see Table  21, that give rise to oligodendrocytes and a type of as trocyte. The very large core protein of NG2 suggests that it may serve functions other than just bearing CS chains.
One such function appears to be the binding of type VI collagen [ 11 I.

ECM and 'soluble' PCs of the CNS
Many PGs can be extracted from the brain using physiological buffers without detergent; others require high salt or denaturing conditions. Although it has been argued that some of these molecules may reside in the cytoplasm of cells, most are probably loosely associated with the ECM.
Most of the PGs in these categories contain CS as their major GAG. Neurocan, a recently cloned CSPG, has a 136 kDa core protein, and contains N 3 CS chains [ 12**]. Its protein sequence places it in a family with aggrecan -the major ECM PG of cartilage -and versican, an ECM PG first found associated with libroblasts. Like these other PGs, neurocan binds the ECM polysaccharide hyaluronic acid via a protein domain that is highly conserved in all three family members. Recent evidence suggests that versican and aggrecan are themselves expressed in the human and chicken brain, respectively [ 130,14*], The Cat-301 antigen is yet another large brain CSPG that binds hyaluronic acid, and immunological evidence suggests that it is related to aggrecan [15*]. One additional hyaluronic acid-binding CSPG, the Tl antigen, has been identified in brain, but at least the hyaluronic acid-binding region of this molecule is apparently unrelated to those of the aggrecan family [ 16-e,17*]. Still other brain CSPGs have been identified with monoclonal antibodies, and remain to be fully characterized [ 14~,18,19=]. autoradiograms. Glypican expression is found throughout the embryo, but is particularly strong in the ventricular zones of the developing CNS. In contrast, cerebroglycan mRNA, which is found only in neural tissue, is not detected in ventricular zones but is found in the layers of immature neurons that form around those zones. In the adult brain, glypican IS expressed by subpopulations of neurons, whereas cerebroglycan is absent. fb-forebrain; rnb-midbrain; hb-hindbrain; tg-trigeminal ganglion.
The distributions of these CSPGs vary from remarkably uniform throughout the brain (PG Tl) to remarkably cell type-and developmental stage-specific. For example, Cat-301 appears around certain subsets of neurons only after activity-dependent critical periods in their development [ 201. Another is transiently expressed during axon outgrowth by several types of neurons involved in the cerebellar mossy fiber system [19-l, As information on the distribution of these CSPG and CS/KSPG core proteins accumulates, so has information on the distribution of different types of CS and KS chains. Several investigators have observed remarkable cell-type specificity in the binding of anti-CS and anti-KS monoclonal antibodies to brain sections (e.g. [ 21,221). During cerebral cortex development, CS is found in the early proliferative neuroepithelium, then later in the marginal zone and subplate regions [23]. With the exception of the subplate, many of the locations of CS expression during development correlate with sites where axons do not grow. For example, CS, as well as KS, are strongly expressed in the roof plate of the spinal cord [24,25].
Recently, a report of an HSPG in brain ECM appeared [26-l. ?his molecule is found in basal laminae outside of and surrounding the chicken brain, but is also expressed transiently in many developing CNS axon tracts. The core protein size (250 kDa) and basal laminar distribution of this PG are reminiscent of perlecan, a major basement membrane PG, but perlecan itself is not found in CNS axon tracts.

Synaptic vesicle PCs
It has long been known that a PG is a major component of synaptic vesicles isolated from the electric organs of lishes. This PG was recently shown to be a transmem brane KSPG, and appears to be involved in acetylcholine transport into vesicles [27-l Immunochcmical data suggest that this molecule is present in many other types of synaptic vesicles, and might therefore play an important general role in transmitter uptake.

Roles of PCs in the nervous system
Insights into the functions of PGs in the nerous system have come t3y many routes, direct and indirect, and man) of the conclusions are still somewhat pr&minan: I I&h lights of what has heen learned are summarized below.

The functions of a family of growth factors are dependent on PCs
All members of the fibroblast growth factor (FGF) fam ily bind GAGS of the heparin/HS class, and apparently must do so to be biologically active [28,29]. Recent studies support a model in which cell-surface HSPGs bind both FGFs and FGF receptors simultaneously, facilitating their interaction [ 301. It is known that at least three FGFs -FGF-1, -2 and -5 -are expressed in the nervous system and exert trophic effects on several classes of neurons [31-34,35**]. Recently, Nurcombe et al. [35**] have suggested that differences in the type of HS carried by a single core protein can render early neuroepithelial cells selectively responsive either to FGF-1 or to FGF-2. This proposition is supported by evidence in other systems that HS structure can impart specificity to HSPG function (e.g. [36,37*,38,39*1).

The kinetics of action of a family of protease inhibitors are dependent on PCs
The structurally related molecules antithrombin III, heparin cofactor II, and protease nexin I all bind and inactivate certain serine proteases (e.g. thrombin) much more rapidly when appropriate GAGS are present. To a large extent, GAGS act by simultaneously binding both protease and protease inhibitor, confining them to the same locality and thereby facilitating their interaction [38]. Of interest to neurobiologists, protease nexin I is abundantly expressed in the CNS, and is thought to regulate neurite outgrowth and neuronal migration [40].

Cell surface PCs participate in establishing cell-cell and cell-ECM contacts
Although cell surface PGs can apparently be the sole receptors for attachment to certain substrata [37-l, PGs usually facilitate interactions mediated through other receptors, such as integrin-dependent cell attachment to ECM molecules [41], and neural cell adhesion molecule (NCAM)-dependent cell-cell adhesion [42,43]. A recent study suggests that cell surface HSPGs are especially important for the interaction of neural cells with fibronectin [44*]. As ECM and cell adhesion molecules are thought to provide important navigational cues to growing axons, the involvement of PGs with such molecules suggests a potential role for PGs in axon guidance. Recent studies in insects support this idea [45**],

ECM PCs regulate cell-cell and cell-matrix interactions
The core protein of at least one PG, perlecan, supports integrin-mediated cell attachment [ 461. In contrast, several PGs inhibit the biological activities of ECM and cell adhesion molecules, at least in vitro. For example, adsorbed CSPCs or CS/KSPGs can render culture substrata inhospitable for neurite growth (24,251. Soluble CSPGs from rat brain also inhibit neurite outgrowth by ~Cl2 cells [47]. Neurocan and the 3F8 CSPG of rat brain (but not aggrecan) inhibit homophilic NCAM and neuron-glial cell adhesion molecule (NgCAM)-binding [489]. A HSPG released by Schwannoma cells specifically blocks the neurite outgrowth-promoting activity of laminin [49]. In some of these cases, the GAG chains of the PGs are required for these actions [ 24,25,49] ; in others they are not [47,48*]. It is not yet known whether these phenomena are direct actions of PGs on neurons, or reflect effects of PGs on the physical characteristics of the culture substratum, so caution must be used in extrapolating these results to in vivo settings, Nonetheless, the distributions of some CSPGs are consistent with a 'barrier' function in vivo (see above). For example, in the developing retina a receding wave of CS expression marks a front of centripetally directed axons, suggesting that axons are guided by their avoidance of CS. Intriguingly, a CSdegrading enzyme disrupts the timing and direction of retinofugal axons in the developing rat retina [50**].

PGs bind virtually every major ECM component.
In addition, molecules such as growth factors (e.g. FGFs) and enzymes (e.g. synaptic acetylcholinesterase) are often immobilized in ECMs through interactions with HSPGs [ 1,511. The importance of PGs in ECM structure and function is illustrated by a muscle cell line that is defective in GAG biosynthesis [52-l. This cell line produces an abnormal basal lamina and, probably as a consequence, fails to form acetylcholine receptor clusters. The cells also fail to form such clusters in response to agrin, a GAG-binding ECM molecule that potently induces receptor clusters on normal muscle cells, and is thought to be involved in synaptogenesis in zuko.

Conclusions
Although much still needs to be learned about nervous system PGs, the identities of many of the major species in the brain are now known. Tracking down the functions of these molecules will probably not be easy. Their biological activities are likely to reside in their capacity to regulate, possibly in subtle ways, the functions of the molecules they bind. Moreover, the repertoire of molecules they bind will probably depend in part on the precise structures of their GAG chains, structures which defy easy analysis. Nevertheless, PGs are likely to continue to receive increasing attention in neurobiology, as their in viao distributions and in vitro activities suggest that they are widely involved in nervous system development and function.  1992, 267:1953619547. The cloning of neurocan is described The cDNA that was obtained ens codes a protein similar to aggrecan and versican, including an amino terminal hyaluronate binding domain, and carbOx)-terminal epidermal growth factor (EGF)-like, lectin-like and complement regulatory-like domains. Evidence is presented that the adult brain contains only a trunk cated form of neurocan, lacking the amino-terminal region.
Isolation of a Large Aggregating Proteoglycan from Human Brain. J Bid Chem 1992, 267:23883-23887. Versican is isolated from human brain and shown to bind hyaluronic acid. The relationship of versican to GHAP @aI hyaluronic acid hinding protein) is also discussed. GHAP, a previously characterized protein, appears to he an amino-terminal fragment of versican. 14.
and  Commun 1992, 188:39WOl. teoglycans in Embryonic Chick Brain. J Biol Chem 1992, 267:12149-12161. One of only a few studies to look at PGs in the avian brain. Aggrecan and its mRNA are found to be transiently expressed in the emhtyonic chick brain. Unlike cartilage aggrecan, the brain form contains only CS and not KS. Also detected in chick brain was a PG with a similar core size, hut that can he distinguished from aggrecan by the presence of both CS and KS chains, reactivity with the HNK-1 monoclonal antibody, and lack of developmental regulation. The Cat-301 immunoreactive CSPG of brain is shown to have properties similar to those of aggrecan. Also, authentic aggrecan from cartilage is shown to react with Cat-301, as well as to another monoclonal antibody that recognizes the same brain PG as Cat-301. Nevertheless, the Cat-301 antigen can he distinguished from aggrecan by virtue of containing few, if any, KS chains and having a lower buoyant density (indicative of a lower carbohydrate/protein ratio) than aggrecan. The data suggest that Cat-301 is either a modified form of aggrecan, or a new member of the aggrecan family.
can Has the Characteristics of a General Extracellular Matrix Component of Adult Brain. J Neurosci 1993, 13:195-207.
The Tl antigen is a large CSPG that can only he extracted from rat brain with chaotropic agents (e.g. guanidine). It appears to be expressed in all parts of the CNS, and is therefore proposed to he a general ECM component. It fails to react with an antiserum directed against a 15 amino acid sequence that is present in the hyaluronic acid-binding domain of aggrecan, is 100% conserved in versican, and 80% conserved in neurocan. This suggests that the Tl antigen may not belong to the aggrecan family. Although this molecule exhibits biochemical properties similar to those of perlecan, its expression in CNS fiber tracts indicates that is not iden tical to perlecan. Until this report, it was not clear that any HSPGs were present in the ECM of the brain. Interestingly, like several other ECM molecules (e.g. laminin and tibronectin), expression of this PG m the parenchyma of the brain appears to be transient. Science 1993, 260:103-106. In this provocative study, an HSPG was isolated from mouse neuron epithelial cells at two stages of development, and tested for binding to FGF-1 and FGF~2. HSPG from embtyonic day 9 cells preferentialb bound FGF-2, whereas HSPG from day 11 cells preferentially bound FGF~l. Similar speciIicity was seen in the ability of the HSPG samples to potentiate the actions of FGF-1 and FGF-2. The developmental switch in binding coincides with a switch in expression of FGFs in the neuroepithelium, from FGF-2 to FGF~l, over the same period.