Neural Tissue Homeostasis and Repair Is Regulated via CS and DS Proteoglycan Motifs

Chondroitin sulfate (CS) is the most abundant and widely distributed glycosaminoglycan (GAG) in the human body. As a component of proteoglycans (PGs) it has numerous roles in matrix stabilization and cellular regulation. This chapter highlights the roles of CS and CS-PGs in the central and peripheral nervous systems (CNS/PNS). CS has specific cell regulatory roles that control tissue function and homeostasis. The CNS/PNS contains a diverse range of CS-PGs which direct the development of embryonic neural axonal networks, and the responses of neural cell populations in mature tissues to traumatic injury. Following brain trauma and spinal cord injury, a stabilizing CS-PG-rich scar tissue is laid down at the defect site to protect neural tissues, which are amongst the softest tissues of the human body. Unfortunately, the CS concentrated in gliotic scars also inhibits neural outgrowth and functional recovery. CS has well known inhibitory properties over neural behavior, and animal models of CNS/PNS injury have demonstrated that selective degradation of CS using chondroitinase improves neuronal functional recovery. CS-PGs are present diffusely in the CNS but also form denser regions of extracellular matrix termed perineuronal nets which surround neurons. Hyaluronan is immobilized in hyalectan CS-PG aggregates in these perineural structures, which provide neural protection, synapse, and neural plasticity, and have roles in memory and cognitive learning. Despite the generally inhibitory cues delivered by CS-A and CS-C, some CS-PGs containing highly charged CS disaccharides (CS-D, CS-E) or dermatan sulfate (DS) disaccharides that promote neural outgrowth and functional recovery. CS/DS thus has varied cell regulatory properties and structural ECM supportive roles in the CNS/PNS depending on the glycoform present and its location in tissue niches and specific cellular contexts. Studies on the fruit fly, Drosophila melanogaster and the nematode Caenorhabditis elegans have provided insightful information on neural interconnectivity and the role of the ECM and its PGs in neural development and in tissue morphogenesis in a whole organism environment.


INTRODUCTION
This chapter highlights the roles of chondroitin sulfate (CS) and dermatan sulfate (DS)-proteoglycans (PGs) in neural biology, heparan sulfate (HS)-PGs were outside the scope of this review and thus are only briefly touched on. However many excellent reviews exist on HS-PGs and their interactions with extracellular matrix (ECM) components in neural development, neural function and potential in neural repair biology (Condomitti and de Wit, 2018;Zhang P. et al., 2018;Roppongi et al., 2020;Xiong et al., 2020;Kamimura and Maeda, 2021;Sakamoto et al., 2021). Roles for HS-PGs in model developmental organisms such as Drosophila melanogaster and Caenorhabditis elegans have also been reviewed (Díaz-Balzac et al., 2014;Blanchette et al., 2017;Kamimura and Maeda, 2017;Saied-Santiago et al., 2017) and the interested reader is referred to these excellent publications for further information.
With the identification of the multiple molecular determinants that provide neuronal connectivity, and with new insights into the modulatory extracellular information regulating axon guidance, neural network and synapse formation, a better understanding of the complexity that neurons face in a living organism is beginning to emerge. Attention is now returning to an ancient regulator of cell-cell interaction: the ECM (Dityatev and Schachner, 2006;Dityatev et al., 2010;Miyata and Kitagawa, 2017;Nicholson and Hrabětová, 2017;Ferrer-Ferrer and Dityatev, 2018;Quraishe et al., 2018;Cope and Gould, 2019;Long and Huttner, 2019;Chelyshev et al., 2020;Jain et al., 2020;Wilson et al., 2020;Carulli and Verhaagen, 2021;Kamimura and Maeda, 2021;Shabani et al., 2021;Su et al., 2021). Among the many matrix components that influence neuronal connectivity, recent studies on the CS-PGs and HS-PGs indicate these ancient molecules form dynamic scaffolds that not only provide a protective environment around cells but are also a source of directive cues that modulate neuronal behavior and synaptic plasticity in tissue development (Haylock-Jacobs et al., 2011;Karamanos et al., 2018;Hayes and Melrose, 2020a;Shabani et al., 2021).

Roles of GAGs in the Evolution of Life and Electrochemical Properties of Tissues
Glycosaminoglycans (GAGs) and PGs are ancient molecules that evolved over a 500 million year period of invertebrate and vertebrate evolution . Natural selection processes 'chose' GAGs with molecular recognition, information storage and transfer properties. The PGs that populated the glycocalyx surrounding cells thus had cell instructive properties through their GAG side chains that interacted with morphogens, growth factors, cytokines, cell receptors, cell adhesion molecules and neurotrophic peptides facilitating regulatory roles in embryonic neural development. GAGs also have electro-chemical properties through their sulfate and carboxyl groups that are ionized at physiological pH. GAG-electroconductive gels in the sensory pores of the skin of elasmobranch fish species (i.e., sharks, rays, and skates) have the capacity to detect protons produced by the muscular activity of prey fish species and this equips them with the ability to undergo electro-location to detect prey species, even under highly turbid water conditions where these are not visible (Bellono et al., 2017). Such gels have ultrasensitive proton detection capability, this information is transferred to a sensory nerve interface in the skin pores and then to the brain stem for signal interpretation.
All GAGs have proton detection capability (Josberger et al., 2016;Selberg et al., 2019) and are ancient molecules that were present during the early stages of the evolution of life . It has been proposed that proton electrochemical ion gradients across membranes drove cellular metabolism and energy production during early evolution (Lane, 2017). In prokaryotic evolution, GAGs were mainly unsulfated or poorly sulfated; however, when eukaryotic cells evolved, sulfated GAGs predominated. Evolution of membrane polarization became possible in eukaryotic cells and membrane energetics emerged (Wilson and Lin, 1980;Niven and Laughlin, 2008;Dibrova et al., 2012;Lynch and Marinov, 2017). Membrane polarization involves the controlled movement of ions across cell membranes, GAGs had fundamental roles to play in these processes through their proton binding properties. All cells in multicellular organisms utilize membrane polarization when undergoing cell signaling, adhesion, proliferation, migration, and cytokinesis. Some cells such as neurons have developed electrical processes to a high level of precision, and this is the basis of the generation of electrical impulses in neural networks that remotely control cells and tissues in higher animals. Further eukaryotic evolution resulted in the development of a glycocalyx around cells. This contained PGs containing GAG side chains with the ability to instruct cellular behavior. The development of pericellular and extracellular matrices populated by PGs facilitated the development of tissues with variable biophysical properties due to these PGs and their co-operative interactions with structural proteins thus driving specialization with the ECM. Neural networks subsequently evolved to control these tissues of increasing complexity. Neurons are highly energetic cell types that utilize Na(+)/K(+)-ATPase pumps to generate energy. This process also generates chemical and electrical gradients across cell membranes. This membrane polarization process is essential for cell signaling and is aberrantly controlled in neurological diseases. Examination of the ECM PGs that control these neural processes has uncovered valuable therapeutic targets (Soleman et al., 2013;Maćkowiak et al., 2014;De Luca and Papa, 2016;Miyata and Kitagawa, 2016;Yang, 2020;Dityatev et al., 2021).
Glycosaminoglycan were fundamental entities in the formation and regulation of neural networks and tissues and the control of cell behavior during morphogenesis, tissue development and in ECM remodeling in tissue repair (Melrose, 2019b). The sulfation patterns of GAGs have roles in cellular molecular recognition and the regulation of physiological processes (Melrose, 2019b). GAG sulfotransferases and glycosyl transferases in progenitor/stem cell niches support the assembly of GAGs of diverse structure and sulfation patterns and are important in the development of pluripotent stem cell lineages with migratory properties (Stanley, 2016). This allows these cells to participate in tissue development and tissue repair.
Sulfate groups are important functional determinants on GAGs. Knock-out sulfotransferase and glycosyl transferase mice have demonstrated the important functional roles of GAGs in tissues (Stanley, 2016). Variable sulfation positions and densities on GAGs convey a range of functional attributes to tissues including an ability to act as electrical conduits to the cell. Sulfate groups are relatively bulky space-filling entities on GAGs, it was pertinent that all spatial orientations and permutations were explored during natural evolutionary selection processes to select GAGs with optimal interactivity. Natural selection forces thus explored many permutations of GAG structural form to optimize cell regulatory capability. Sulfate groups convey interactive molecular recognition and information transfer capability to GAGs and their interactions with growth factors, receptors, morphogens, ECM components, proteases, and protease inhibitory proteins regulate cell signaling processes in tissue morphogenesis and skeletogenesis. Knockout of glycosyl transferases, that are required for GAG assembly, has produced GAG-deficient mice (Stanley, 2016) that have allowed examination of the roles GAGs play in tissue form and function and how these regulate physiological processes in health and disease. The inherent charge transfer and storage properties of GAGs is a "glycocode" that provides sophisticated cell instructive information (Gabius, 2018;Kaltner et al., 2019).

CNS/PNS ECM PGs/GAGs, Cellular Regulation, and Neural Tissue Development
As already discussed, GAGs have electrochemical properties equipping them with cell regulatory abilities (Lane, 2017;Selberg et al., 2019). At the individual cell level, voltage gradients occur across cell membranes as so-called, action potentials (Strbak et al., 2016) which form part of the cell signaling and communication machinery of cells, i.e., membrane polarizationdepolarization underlying the generation of electrical signaling in neural networks. Proton conductivity is important in many natural cellular processes including oxidative phosphorylation in mitochondria and energy production, uncoupling of membrane potentials during membrane polarization-de-polarization and neural potentiation, as well as in the priming of cells for proliferative events, apoptosis or cell migration (Wilson and Lin, 1980;Vellai et al., 1998;Niven and Laughlin, 2008;Dibrova et al., 2012;Lynch and Marinov, 2017). Electrochemical reactions control cell and tissue polarity and regulate cell behavior, ECM PGs facilitate electrocommunication between cells and their extracellular microenvironments. Cells sense changes in their microenvironments through micromechanical and electrochemical cues from the ECM allowing the cell to maintain a homeostatic tissue compositional balance thus providing optimal tissue functional properties (Guilak et al., 2021;Melrose et al., 2021). GAGs can detect proton gradients and are electroconductive entities that participate in microelectronic events during membrane polarization forming the basis of cell signaling (Wilson and Lin, 1980;Vellai et al., 1998;Niven and Laughlin, 2008;Dibrova et al., 2012;Lynch and Marinov, 2017). Neurons are particularly sensitive to electrostimulation in microelectronic events leading to polarization of the activated neuron cell membrane, however, membrane polarization occurs in all cells to some extent and is the basis of cell signaling during cellular attachment, migration and transmission of signals from cell to cell not only during development but also in neural repair and functional nerve recovery from trauma (Hortobágyi and Maffiuletti, 2011;Hayes and Melrose, 2020b). The GAG components of PGs participate in neurotrophic regulation of cellular movement in the development of neural networks and also in neural repair processes. A diverse collection of neuroregulatory molecules participate in these processes guided by cues from ECM PGs, which are discussed later in this chapter.

THE CHONDROITIN SULFATE AND DERMATAN SULFATE COMPONENTS OF NEURAL PROTEOGLYCANS
Chondroitin sulfate is the most abundant GAG of the human body and CS side chains are found on a diverse range of PGs. CS is a linear GAG consisting of D-glucuronic acid glycosidically linked to N-acetyl galactosamine to form repeat disaccharides assembled into CS side chains (Zhang, 2010) on PGs up to ∼20 kDa in size (Figure 1). D-glucuronic acid also undergoes epimerization and inversion in structure to form L-iduronic acid in the related GAG, DS also known as CS-B (Figure 2).
Chondroitin sulfate is O-sulfated at C-4 or C-6 of the GalNAc, whereas in DS GalNAc is almost exclusively 4-Osulfated and minor proportions of L-idoA may be O-sulfated at C-2 (Malmström et al., 2012). The conversion of GlcA into IdoA is variable ranging from one to almost 99% conversion of GlcA to IdoA (Malmström et al., 2012). IdoA residues are not regularly distributed along the CS/DS chain and occur in blocks of ≥6 IdoA residues, alternating IdoA/GlcA units, or as isolated IdoA units interspersed within stretches of unmodified GlcA (Tykesson et al., 2018). DS epimerase-1 and dermatan 4-Osulfotransferase-1 form complexes that generate long epimerized 4-O-sulfated blocks. The presence of idoA in CS/DS alters its properties since a more flexible chain is generated that is more able to explore spatial orientations that maximize binding opportunities with prospective ligands (Ferro et al., 1990). IdoA substituted CS/DS influences cellular properties, such as migration, proliferation, differentiation, angiogenesis and regulates cytokine/growth factor activities (Thelin et al., 2013). CS and DS occur in significant amounts in the brain and have important roles to play in CNS development. DS sulfate epimerase 2 (DS-epi2) is ubiquitously expressed in the infant brain whereas DS epimerase 1 (DS-epi1) expression is faint at all developmental stages (Akatsu et al., 2011). DS-epi2 but not DS-epi1 plays dominant roles in the epimerization of CS/DS and has crucial roles to play in postnatal brain development. CS/DS hybrid chains have roles in the development of the cerebellum with the expression of crucial disulfated CS/DS disaccharides spatiotemporally regulated by specific sulfotransferase enzymes (Mitsunaga et al., 2006). Ubiquitous expression of chondroitin 4-O-sulfotransferase-1 (C4ST-1) and C4ST-2 in the postnatal mouse brain contrasts with dermatan 4-O-sulfotransferase-1 (D4ST-1) and uronyl 2-sulfotransferase FIGURE 1 | Structure of the CS/DS disaccharides (A) and nomenclature of the CS and DS disaccharide glycoforms (B) as proposed by Malavaki et al. (2008).
(UST) expression which are restricted to the developing cerebellum. The proportions of DS-specific, 4-sulfated IdoA-GalNAc (iA) and 2-sulfated IdoA-GalNAc (iB) produced by sequential D4ST-1 and UST activity has been shown to be highest in CS/DS chains isolated from the developing cerebellum with a 10-fold increase in iB evident. GlcA/IdoA(2-O-sulfate)-GalNAc(6-O-sulfate) (D/iD) and GlcA/IdoA-GalNAc (4,6-Odisulfate) (E/iE) levels, however, decrease to 50 and 30%, respectively, in the developing cerebellum. Thus IdoA-containing iA and iB and D/iD and E/iE units in CS/DS hybrid chains both have important roles to play in the development of the cerebellum and postnatal brain development. The diverse structures that are possible with CS provide multifunctional properties to CS-PGs (Abbott and Nigussie, 2020), with dynamic changes in CS structure providing adaptable regulatory properties to PGs in tissue development and in pathological conditions (Galtrey and Fawcett, 2007). CS-PGs as components of perineuronal nets (PNNs) have neuroprotective properties and regulate neural plasticity and cognitive learning through specific CS mediated interactions (Dyck and Karimi-Abdolrezaee, 2015). PGs are ubiquitous secreted ECM components (Figure 3) that also occur attached to cell surfaces either as transmembrane or glycophosphatidylinositol (GPI)-anchored structures, and intracellularly as granular deposits in some cells (Figures 4, 5). Perlecan is referred to as a HS-PG, however, in many tissues it is a hybrid CS/HS PG and is thus included in this review, particularly in view of its many interesting properties in neural tissues. The chain length of CS, 3D spatial presentation and density of its sulfate groups control its physicochemical and cell interactive and biological properties in tissues through interactions with a diverse range of ligands that regulate many physiological processes (Galtrey and Fawcett, 2007;Dyck and Karimi-Abdolrezaee, 2015). GAGs represent major ECM components of the brain, constituting up to 60% of its mass during early embryonic development and 20% in the adult central nervous system/peripheral nervous system (CNS/PNS). CS substituted PGs are one of the most abundant components of the CNS/PNS. HA is also a major component. HA has a simple structure and is the only non-sulfated GAG, but nevertheless has important biophysical properties which are important in the hydration and compartmentalization of the CNS/PNS. High molecular weight HA is anti-inflammatory, minimizes neuroinflammation and exhibits cell interactive properties that regulate cellular migration, proliferation and differentiation (Sherman et al., 2015). HA is also a component of the sub-ventricular and subgranular dentate gyrus of the hippocampus which are two regions of the brain containing neuroprogenitor stem cell populations in niches known as fractones Mercier, 2016;Sato et al., 2019).

DEVELOPMENTAL ANIMAL MODELS USED TO EXAMINE NEURAL DEVELOPMENT AND REGULATION IN A WHOLE ORGANISM ENVIRONMENT
Studies on the fruit fly, D. melanogaster (FitzGerald et al., 2006;Nishihara, 2010;Losada-Perez et al., 2016;Davis et al., 2019) and the nematode C. elegans (White et al., 1976;Mulcahy et al., 2018;Schafer, 2018;Kovács et al., 2020;White, 2020) have provided insightful information on the role of the ECM and some FIGURE 2 | The biosynthesis of CS and DS chains showing their diverse disaccharide glycoforms that are functional in the juvenile and adult CNS/PNS and during the development and repair of the CNS/PNS. Biosynthesis of the tetrasaccharide linker sequence by addition of a xylose residue to a serine residue in the proteoglycan core protein followed by addition of two Galactose and a GlcA residue (a). Initiation of CS chain elongation occurs by addition of a GalNAc residue by chondroitin N-acetylgalactosaminyltransferase-1 or chondroitinpolymerase (b). Elongation of the CS chain occurs by sequential additions of GlcA and GalNAc to the nascent non-reducing terminus by chondroitin polymerases (c). The CS chain is sulfated by chondroitin-4 and 6-sulphotransferases, or the GlcA residue of the O-disaccharide unit is epimerized to IdoA with inversion in structure from a β-D conformation to an α-L conformation followed by a series of sulphotransferases and uronyl-2-sulphotransferase to form the CS-A, CS-B, CS-C, CS-D, CS-E and DS-iA, DS iB, CS-iC, DS-iD, and DS-iE isoforms as shown (d-f). The 4-O-sulfation pathway is most active in the adult brain (d) while various DS isoforms regulate brain development and repair processes (e). The 6-sulfation pathway (f) is most active in the juvenile brain. The DS-iO and DS-iC units have yet to be confirmed. Figure modified from Malmström et al. (2012) and Miyata and Kitagawa (2017).
Frontiers in Cell and Developmental Biology | www.frontiersin.org of its specific components in neural development and tissue morphogenesis in a whole organism environment. White et al. (1986) undertook the first complete mapping study of the nematode's nervous system using manual reconstruction of serial electron micrographs, to characterize the morphology of each of the 302 neurons in the adult nematode and their interconnected chemical and electrical synapses. This study is an invaluable guide on the C. elegans neural network and has significantly influenced studies on behavioral neurobiology and network science. The nervous system of C. elegans has a total of 302 invariantly organized neurons, that have been grouped into 118 categories. Neurons in C. elegans have simple morphologies displaying few, if any, branches and are generally highly connected through local synaptic connections with neighboring neurons, ∼ 5000 chemical synapses, 2000 neuromuscular junctions and 600 gap junctions have been identified in C. elegans. The specific patterns and functional properties of electrical synapses of the C. elegans nervous system FIGURE 4 | Composite schematic depicting the structural organization of cell membrane attached CS-proteoglycans in the CNS/PNS. Phosphacan precursor, receptor protein tyrosine phosphatase (RPTP)-1A and 1B forms (A,B) and soluble phosphacan released by proteolysis of (A) as shown (C). A non-glycanated truncated phosphacan variant has also been described (D). Syndecan-1 (SDC-1) (E) and syndecan-3 (SDC-3) (F). The transmembrane form of NG2 proteoglycan (CSPG4) (G) and its soluble form released from cells by protease cleavage close to the cell membrane (H). The soluble form of CSPG4 becomes lodged in the ECM through interactions mediated by its LamG N-terminal motifs and by interactions of its central cysteine-rich domain with type IV and VI collagen. Thrombomodulin showing its terminal lectin-like domain, six EGF repeat modules, thrombin binding region which allows it to act as an anti-coagulant in the brain microvasculature and transmembrane cell attachment domain (I). Appican (J), neuroglycan-C (K), and the HA-receptor CD-44 (L) with protease cleavage sites that results in the generation of soluble forms of these proteoglycans. The CS-E chains of appican bind strongly to the growth factors midkine and pleiotrophin, neuroglycan-C binds to ephrin cell surface receptors resulting in induction of cell signaling mediated by the ephrin cytoplasmic regions while sCD44 binds to HA in the ECM through its disulphide stabilized A, A and Ig folds. Several CAMs also act as CS-PG receptors through interaction with the CS side chains of these PGs (see Figure 7). have been systematically examined through a genome-and nervous-system-wide analysis of the expression patterns of the invertebrate electrical synapse constituents, the innexins (Hu, 2007;Güiza et al., 2018;Bhattacharya et al., 2019). Innexins are transmembrane proteins that form gap junctions in invertebrates. Highly complex expression patterns are evident throughout the nervous system, and when animals enter an insulindependent arrest stage due to exposure to harsh environmental FIGURE 5 | Schematic depictions of the structural organization of the transmembrane CS-PG receptors CD47 showing its interactions with macrophages, integrins and thrombospondin-1 (TSP-1) (A), betaglycan dimer (B) and endocan (C). Betaglycan forms an active dimer on the cell surface. Protease cleavage sites are indicated which release fragments of the betaglycan ecto-domain. Endocan has an EGF-like and a phenylalanine rich domain and a single CS/DS GAG chain attached to its C-terminus.
conditions, termed the dauer stage, C. elegans larvae undergo Dauer arrest at the second molt (Cassada and Russell, 1975). Many insights into the signaling pathways and the molecular mechanisms that govern this developmental transition have been uncovered in the last decade. Dauer pheromone the major physiologic signal that promotes Dauer arrest, has been purified, identified, and synthesized and the vast majority of Dauer regulatory genes are now known (Jeong et al., 2005). Dauer pheromone (daumone, ascr#1) is the first C. elegans pheromone identified. Electrical synapse remodeling is responsible for the altered locomotory and chemosensory behavior of Dauer affected neurons (Starich et al., 2009). These neurons are regulated by a mechanism that involves FoxO transcription factors that mediate dynamic innexin expression plasticity in a neurontype-and environment-specific manner (Hobert, 2010;Amran et al., 2021). C. elegans is a useful model for the examination of developmental processes regulated by ECM components (Hobert, 2010;Amran et al., 2021). HSPGs and chondroitin proteoglycans (CPGs) both have prominent roles to play in developmental processes in C. elegans (Saied-Santiago et al., 2017). Most ECM molecules in C. elegans are conserved and are homologues to mammalian proteins, however, fewer ECM protein isoforms are present and these exhibit less redundancy compared to in mammalian tissues. Furthermore, mutations in C. elegans produce comparable developmental defects to those evident in mammals. The small size, short lifespan of 3-4 days and defined neural interconnectivity of the C. elegans nervous system facilitate its use in systematic functional studies that have yielded valuable insights into neuronal differentiation (Chisholm and Jin, 2005). This information is also relevant to more complex neural systems since the basic cellular machinery of synaptic transmission is highly conserved across species. In C. elegans, 719 out of ∼20,000 genes (∼4%) of its genome encode matrisome proteins, including 181 collagens, 35 glycoproteins, 10 proteoglycans, and 493 matrisome-associated proteins, 173 out of the 181 collagen genes are unique to nematodes and are predicted to encode cuticular collagens (Teuscher et al., 2019).
Comparative genomic studies on human and C. elegans has identified orthologous genes in C. elegans that have comparable regulatory properties to human genes and demonstrated mechanisms relevant to human biology. C. elegans represents an experimental system that can be used in genetic approaches to address biological questions relevant to human development, physiology, and disease. Genetic based phenotype screens have identified C. elegans genes homologous to human diseaseassociated genes, and fundamental properties about their roles and mechanisms of action in pathological human tissues. Reverse genetic methods developed in C. elegans have expanded the repertoire of genetic approaches to examine the roles of specific genes in human disease processes. These methods have the ability to phenocopy loss-of-function mutations by feeding worms bacteria expressing double-stranded RNA, RNAi. Studies in C. elegans have also facilitated genome-wide screens or screens specifically targeting human disease genes and the large-scale generation of deletion or point mutations for functional genetic studies (Kim et al., 2018).
The Drosophila matrisome consists of 641 genes, 27 are homologs/orthologs to human core matrisome genes, 219 are homologs/orthologs to mammalian matrisome genes and the remaining 395 are genes are specific to Drosophila . Comparative genomic analyses have been undertaken between human and Drosophila genomes and core promoter regions. Although fruit flies have a genome that is 25 times smaller than the human genome, many fruit fly genes have comparable genes in humans that control the same biological functions. Twelve fruit fly genome sequences are available in the FlyBase database 1 , a collaboration of Harvard, Cambridge, Mass.; Indiana, Bloomington; and the University of Cambridge, United Kingdom. The fruit fly genome sequences are also available from NIH's National Center for Biotechnology Information 2 , European Molecular Biology laboratory's Nucleotide Sequence Database, EMBL-Bank 3 , and DNA Data Bank of Japan, DDBJ 4 (FitzGerald et al., 2006).
As found in higher organisms, PGs have important roles in cellular regulation critical to the development of metazoan organisms. C. elegans, however, produces a large amount of non-sulfated chondroitin (Chn) in addition to a small amount of low sulfation CS. Until recently, C. elegans was known to express nine Chn/CS-PGs dissimilar to vertebrate CS-PGs. A recent glycoproteomic study identified 15 additional CPGs in C. elegans and three of these were homologous to human proteins, thus selected CS-PG core proteins appear conserved throughout evolution (Noborn and Larson, 2021). Bioinformatic analysis of primary amino acid sequence data identified a broad range of functional domains in these C. elegans PGs, thus specific PG core protein mediated functional properties appeared to have evolved early in metazoan evolution.
Drosophila melanogaster has also proved to be a useful model for the investigation of developmental GAG functions in vivo confirming in vitro findings. HS and CS GAG side chains of PGs are structurally conserved between Drosophila and mammals, including humans. Mutant and RNAi fruit flies show that HS-PGs and CS-PGs play key roles in the regulation of developmental signaling pathways involving FGF, Wingless (Wg)/Wnt, Hedgehog, and Decapentaplegic (Dpp, a ligand of the TGFβ superfamily). Glycosyl transferases, sulfotransferases, sugar-nucleotide transporters including 3 -phosphoadenosine 5phosphosulfate (PAPS) transporters, all have important roles to play in GAG biosynthesis and the functional status of PGs in neuronal development and maintenance (Nishihara, 2010). It should be noted, however, that the major non-sulfated Chn in C. elegans has crucial roles in embryonic cell and tissue development and tissue morphogenesis. Since Chn is present on mammalian PG core proteins along with CS no studies have been possible to specifically target the functional roles of Chn in these PGs, however, insights into the biological properties of Chn in mammals can be gleaned from the use of Chn as a drug in the treatment of osteoarthritis (OA) (Singh et al., 2015). A meta-analysis of 43 reviews which analyzed the use of Chn for the treatment of OA and alleviation of joint pain yielded moderate to inconclusive results. However, Chn elicited a significant improvement in the anti-inflammatory profile of synoviocytes and chondrocytes in an OA model analyzed by multiplex and Western blot analysis. Chn significantly decreased the levels of several pro-inflammatory cytokines 6,7,9,15,17), anti-inflammatory cytokines (IL-4, IL-10), chemokines (IL-8, MCAF, MIP-1a, MIP-1b, RANTES) in synovial fluid samples and decreased expression of the OA biomarkers MyD88 and MMP-13 (Vassallo et al., 2021). Evidence therefore exists that Chn displays anti-inflammatory properties in mammalian tissues. However in C. elegans where Chn predominates over CS the biological properties of Chn have more profound effects on neural biology. A number of studies have demonstrated the fundamental biological roles played by Chn, CS, and DS, attached to the core proteins of cell surface and ECM PGs . PGs decorated with CS, DS, or HS have diverse roles in growth factor, morphogen and cytokine-mediated cell signaling through cellular receptors that play critical roles in the development of the CNS . As discussed later in this chapter, these functions of PGs are closely associated with GAG sulfation patterns. Surprisingly, non-sulfated Chn is indispensable in the morphogenesis and cell division of C. elegans, as revealed by RNA interference experiments of the recently cloned chondroitin synthase gene and by the analysis of squashed vulva (sqv) gene mutants. It should be noted that while orthologous forms of human perlecan/HS-PG2 exist in C. elegans (UNC-52) and D. melanogaster (Trol) these have a different modular structure to human perlecan and are devoid of domain-I and thus they lack HS substitution (Celestrin et al., 2018). Thus the interactive properties provided by the HS chains of human perlecan do not occur in these orthologs and the interactive properties conveyed by these form of perlecan is due to modular components in their core proteins (Condomitti and de Wit, 2018;Martinez et al., 2018).

The Instructional Properties of CS-and HS-PGs in Neural Development and Tissue Morphogenesis in Caenorhabditis elegans
Blocking of chondroitin synthesis results in cell proliferative defects in early embryogenesis in C. elegans and leads to early embryonic death . Mutations in eight sqv genes in C. elegans causes defects in embryonic cytokinesis and in vulval morphogenesis during postembryonic development. Sqv-1, 2, 3, 4, 6, 7, 8 control CS and HS biosynthesis, while sqv-5 encodes a bifunctional glycosyltransferase responsible for the biosynthesis of Chn, but not HS (Hwang et al., 2003a,b). Sqv mutations in C. elegans have lethal consequences due to disruption in cell proliferation and the lack of the formation of an extracellular space between the egg and the eggshell, apparently due to disruption in the normal GAG containing ECM structures. Cloning and characterization of the sqv-2 and -6 genes showed that sqv-6 encoded a protein similar to human xylosyltransferase, while sqv-2 encoded a protein similar to human galactosyltransferase II. SQV-6 and SQV-2 proteins act in concert with other SQV proteins to catalyze the stepwise formation of the PG core protein linkage tetrasaccharide GlcAβ1,3Galβ1, 3Galβ1,4Xylβ-O-(Ser), common to CS and HS (Hwang et al., 2003a,b). This linkage tetrasaccharide acts as an acceptor molecule for the assembly of the CS and HS chains. Chain elongation is initiated by the addition of GlcNAc or GalNAc, with the former addition resulting in the biosynthesis of HS chains by sequential additions of GlcA and GlcNAc, while if GalNAc is the initial sugar added to the acceptor group this results in the synthesis of CS chains. These GAG chains are sulphated in a later biosynthetic stage at various positions on GlcNAc or GalNAc by specific sulfotransferases. GlcA can also be epimerised to IdoA and sulphated at O-2 in HS chains. HS chains can be sulphated at multiple positions. CS sulfation is an important functional determinant in the regulation mammalian neural tissue development and repair, however, in C. elegans and D. melanogaster the Chn chains are not sulphated but nevertheless have essential roles to play in early embryonic development and tissue morphogenesis in later developmental stages.
Vertebrates produce multiple CS-PGs with important roles in development and the mechanical performance of tissues. The Chn chains in C. elegans are not sulfated, but nevertheless they still play essential roles in embryonic development and vulval morphogenesis (Olson et al., 2006). C. elegans Chn PG core proteins, do not share sequence similarities with PGs from D. melanogaster or Hydra vulgaris. The C. elegans CPG-1 and CPG-2 PGs are expressed during embryonic development and bind chitin, which may have a structural role to play in the egg (Olson et al., 2006). Chitin is a widespread polymer in nature and is a polymer composed of N-Acetyl glucosamine. Chitin is a primary component of fungal cell walls, the exoskeletons of arthropods such as crustaceans and insects and the scales of fish. Depletion of CPG-1/CPG-2 results in multinucleated single-cell embryos in C. elegans, this is also observed with depletion of the SQV-5 chondroitin synthase protein, Chn chains of CPG1/CPG2 play essential roles in cytokinesis. This is achieved through regulation of GAG biosynthetic enzymes. C. elegans microRNA mir-79, an ortholog of mammalian miR-9, controls sugar-chain homeostasis by targeting two proteins in the PG GAG biosynthetic pathway: a chondroitin synthase (SQV-5; squashed vulva-5) and a uridine 5 -diphosphate-sugar transporter (SQV-7). Loss of mir-79 causes neurodevelopmental defects through dysregulation of SQV-5 and SQV-7. This results in a partial shutdown of HS biosynthesis that effects the LON-2/glypican pathway and disrupts neuronal migration. MicroRNA thus represents a regulatory axis that maintains PG homeostasis. Sqv genes 1-8 control the invagination of vulval epithelial cells, normal oocyte formation and embryogenesis. Sequencing of sqv-3, sqv-7, and sqv-8 genes indicated potential roles for the proteins they encode in glycolipid or glycoprotein biosynthesis. sqv-3, -7, and -8 affect the biosynthesis of GAGs and the bioactivity of PGs establishing their essential roles in tissue morphogenesis and pattern development in C. elegans (Bulik et al., 2000).
Sulfation of PG GAG side chains has critical roles to play in the cell regulatory properties of PGs and their roles in many essential physiological processes. Sulfation reactions involves activated sulfate, and the universal sulfate donor 3 -phosphoadenosine 5 -phosphosulfate (PAPS). In animals, PAPS is synthesized from ATP and inorganic sulfate by PAPS synthase, genetic defects in PAPS synthase 2, one of two PAPS synthase isozymes, causes dwarfism. In order to better understand the developmental role of sulfation in tissue PGs, a C. elegans PAPS synthasehomologous gene, pps-1 has been cloned and the depleted expression of its product, PPS-1 examined (Dejima et al., 2006). PPS-1 protein exhibits specific roles in the formation of PAPS in vitro. Disruption of the pps-1 gene by RNAi methods causes widespread developmental tissue defects, a decrease in GAG sulfation in the pps-1 null mutant exhibits larval lethality. Sulfation is essential for normal growth and the integrity of the epidermis in C. elegans and has been used as a model to demonstrate the role of HS modifications in a defined biological process. Genetic analyses suggest that syndecan/sdn-1 and HS 6-O-sulfotransferase, hst-6, function in a signaling pathway and glypican/lon-2 and HS 2-O-sulfotransferase, hst-2, function in a parallel pathway. HS modifications may be part regulated at the level of tissue expression of genes encoding for HS-PGs and HS modifying enzymes. There is a delicate balance in such HS modifications that may deleteriously effect cell migration, HS is a critical regulator of cell signaling in normal development and disease. HS-PGs have roles in the structural organization of neurochemical synapses, involving interactions with the core protein as well as the HS side chains (Cizeron et al., 2021). Specific modifications to HS contribute to a sugar code which provides specificity to synaptic interactions. SDN-1 is a unique C. elegans syndecan ortholog found in synaptic junctions. 3-O sulfation of SDN-1 maintains the ECM protein punctin/MADD-4/ (MAP kinase-activating death domain protein) that defines the synaptic domains, however, in mammals 3-O sulfation is a rare modification in HS despite the seven HS modifying enzymes that can produce 3-O sulfation.
Punctin/MADD-4, a member of the ADAMTSL ECM protein family, is a synaptic organizer in C. elegans. MADD is an enzyme encoded by the MADD gene. The Ig-like domain of MADD is the primary determinant for N-MADD-4B interactions with NLG-1 in vitro (Platsaki et al., 2020). At GABAergic neuromuscular junctions, the short isoform MADD-4B binds the ectodomain of neuroligin (NLG-1), which is also a postsynaptic organizer of inhibitory synapses (Tu et al., 2015). Proteolysis of MADD-4B generates N-MADD-4B, which contains four thrombospondin domains and one Ig-like domain that bind NLG-1 (Maro et al., 2015). A second processing event eliminates the C-terminal Ig-like domain of N-MADD-4B and its ability to bind NLG-1. The death domain of the type 1 tumor necrosis factor receptor (TNFR1) mediates the downstream effects of TNF. MADD interacts with TNFR1 residues and is a component of the TNFR1 signaling complex. The MADD death domain stimulates ERK and c-JUN N-terminal kinase MAP kinases inducing phosphorylation of cytosolic phospholipase A2. Thus, MADD links TNFR1, MAP kinase activation and arachidonic acid release, which may explain the pleiotropic effects of TNF.
Growth cones facilitate the repair of damaged neural tissue by promoting axon regeneration, syndecan, is required for growth cone function during axon regeneration in C. elegans (Edwards and Hammarlund, 2014;Gopal et al., 2016Gopal et al., , 2021. In the absence of syndecan, regenerating growth cones are unstable and they collapse, impeding regrowth to target cells. Syndecan has two distinct functions during axon regeneration: (i) axon guidance requiring its HS-dependent expression outside the nervous system (ii) intrinsic growth cone stabilization mediated by the SDC core protein independently of HS.

The Instructional Properties of CS-and HS-Proteoglycans in Neural Development and Tissue Morphogenesis in D. melanogaster
Blocking Chn synthesis results in defects in cytokinesis and embryogenic development in D. melanogaster leading to early embryonic death. This demonstrates the essential developmental roles Chn plays in Drosophila embryonic cytokinesis and cell division. Drosophila has proved to be a useful model for the investigation of developmental GAG functions in vivo confirming in vitro findings with GAGs (FitzGerald et al., 2006;Nishihara, 2010). HS and CS GAG side chains of PGs are structurally conserved between Drosophila and mammals, including humans. CS sulfation is an important functional determinant in the regulation of mammalian neural tissue development and repair; however, in C. elegans and D. melanogaster the Chn chains are not sulfated but, nevertheless, have essential roles to play in early embryonic development and tissue morphogenesis in later developmental stages.
Windpipe (Wdp) is a novel CS-PG recently identified in Drosophila. Wdp is a single-pass transmembrane protein with leucine-rich repeat (LRR) motifs and has three extracellular CS chain attachment sites (Takemura et al., 2020). Wdp modulates the Hedgehog (Hh) cell signaling pathway. In the wing disk, overexpression of Wdp inhibits Hh signaling an effect that is dependent on its CS chains and protein interactive LRR motifs. The JAK/STAT (Janus Kinase/Signal Transducer and Activator of Transcription) signaling pathway regulates adult stem cell activities and is essential for the maintenance of intestinal homeostasis in Drosophila. Wdp interaction with the receptor Domeless (Dome) promotes its internalization and lysosomal degradation. Wdp thus acts as a negative feedback regulator of JAK/STAT cell signaling and is a novel regulatory component of JAK/STAT signaling in Drosophila adult intestines (Ren et al., 2015).
Glycosaminoglycans such as HS and CS have roles in intercellular signaling thus disruptions of genes encoding enzymes that mediate GAG biosynthesis have severe consequences in Drosophila and mice. Mutations in the Drosophila gene sugarless, encoding a uridine diphosphate (UDP)-glucose dehydrogenase, impairs developmental signaling through the Wnt family member Wingless, and FGF and Hedgehog signaling pathways. Undersulfated and oversulfated CS chains are implicated in neural development, cloning of a chondroitin synthase homolog in Caenorhabditis elegans and depletion in Chn expression results in defects in cytokinesis in early embryogenesis and early embryonic death demonstrating the essential role Chn plays in early developmental processes .
Drosophila has an NG2 homologue called kon-tiki (kon), that promotes CNS repair (Losada-Perez et al., 2016). Crush injury upregulates kon expression and induces glial cell proliferation and differentiation by activating glial genes and prospero (pros). Negative feedback loops with Notch and Pros allow Kon to drive the homeostatic regulation of neuronal repair with modulation of Kon levels in glia, potentially preventing or promoting CNS repair (Losada-Perez et al., 2016). The interplay between Kon, Notch, and Pros is therefore essential in neural repair in Drosophila. Prospero homeobox protein-1 is encoded by the PROX1 gene in humans. This pan-neural protein has essential roles to play in the proper differentiation of neuronal lineages and in the expression of genes in the Drosophila CNS. Prospero is a sequence-specific DNA-binding protein that can act as a transcription factor through interaction with homeodomain proteins to differentially modulate their DNA-binding properties (Hassan et al., 1997;Yousef and Matthews, 2005). Functional interactions between Prospero and homeodomain proteins is supported by observations showing that Prospero, together with the homeodomain protein, Deformed, are required for proper regulation of a Deformed-dependent neural-specific transcriptional enhancer (Hassan et al., 1997). The DNA-binding and homeodomain protein-interactive properties of Prospero are localized in its highly conserved C-terminal region.
Syndecan (Sdc) is a conserved transmembrane HS-PG bearing CS chains on its ectodomain. In vertebrates, this extracellular domain of Sdc is shed and acts as a soluble effector of cellular communication events, and the Sdc cytoplasmic domain participates in intracellular signaling needed to maintain epithelial integrity. In Drosophila, Sdc has been shown to be necessary for Slit signaling-dependent axonal guidance during CNS development (Chanana et al., 2009). Sdc acts in a cell-autonomous manner in Slit-receiving cells and that its membrane-anchored extracellular domain is sufficient to mediate Slit signaling. The HS-PG Dally-like protein (Dlp), which lacks CS on its extracellular domain, can only partially substitute for Sdc function but its activity is not restricted to the Slit target cells. Sdc and Dlp act in a cooperative but non-redundant manner in neural tissues with Dlp transferring Slit from its site of expression to the target cells, where it interacts with CS-modified Sdc.

Caenorhabditis elegans Netrins and Neural Development
UNCoordinated-6 (UNC-6) was the first C. elegans member of the netrin family that was discovered (Krahn et al., 2019). UNC-6 shares homology to human netrin-1, and is a key signaling molecule in the regulation of directional axonal migration in nematodes (Krahn et al., 2019). Similar to netrin-1, UNC-6 interacts with multiple receptors to guide axonal migration (Moore et al., 2007;Rajasekharan and Kennedy, 2009;Ogura et al., 2012). Netrin is a key guidance protein regulating the orientation of axonal growth during neural network formation in C. elegans. LON-2/glypican, modulates UNC-6/netrin signaling through interactions with the UNC-40/DCC (deleted in colorectal carcinoma) receptor (Yang et al., 2014). LON-2 expressed on the cell surface in the intestine and hypodermis in C. elegans and in D. melanogaster promotes growth factor binding in several developmental processes, negatively regulating the TGF-β receptor signaling pathway and BMP-like signaling that regulates tissue growth and body length. N-terminal sequencing of the signal peptide of LON-2, identified a 14 cysteine domain of functional importance, SGXG GAG attachment site and C-terminal GPI anchor site showing that LON-2 is a member of the glypican family (Eisenhaber et al., 2000;Gumienny et al., 2007). The other C. elegans glypican, gpn-1, has no significant effect on the body size (Blanchette et al., 2015). Null mutations in C. elegans genes encoding HS biosynthetic enzymes that process the HS side chains of LON-2, significantly reduce body size. hse-5, hst-2, and hst-6 encode C. elegans counterparts to mammalian glucuronyl C5-epimerase, 2 O-sulfotransferase, and 6 O-sulfotransferase, respectively. This demonstrates the important functional role HS plays in neural development in C. elegans and the importance of the HS sulfation patterns for this activity (Bülow and Hobert, 2004;Gysi et al., 2013;Díaz-Balzac et al., 2014;Saied-Santiago et al., 2017). HS chains of HS-PGs carry multiple structural modifications due to sulfation and epimerization of GlcA that influence their ligand binding properties. This is why HS-PGs have such diverse effects in tissue and axonal development. The core proteins of C. elegans SDN-1 and glypican/LON-2 and HS modifying enzymes thus both have roles in axonal guidance through interactions with UNC-6/Netrin (Rhiner et al., 2005). C. elegans SDN-1/syndecan control of neural migration and axonal guidance also occurs through regulation of Slit/Robo signaling in parallel with C5-epimerase HSE-5, and/or the 2Osulfotransferase HST-2 activity, which provide distinct regulatory HS modification patterns on SDN-1.

MAMMALIAN NEURONAL PROTEOGLYCANS
Neural PGs occur as large and small extracellular, cell surface and intracellular components. The salient features of neural PGs are summarized in Table 1

Roles for the CS-Rich Lectican PG Family in Perineuronal Net Structures
The lectican family of neural PGs have similar structures to aggrecan but do not contain keratan sulfate (KS) or a G2 globular domain. Furthermore, their molecular dimensions are smaller due to shorter core proteins and less extensive distributions of CS side-chains (Yamaguchi, 2000). Lectican PGs occur as diffuse ECM components and as dense PNN structures attached to HA through interactions with lectican N-terminal HA-binding regions. This aggregate is stabilized by tenascin-R and Bral-1 (Hyaluronan and Proteoglycan Link Protein 2; HAPLN2). The form of aggrecan found in brain differs from that of cartilage aggrecan in that it contains less KS chains, and its CS chains are less densely distributed along the CS1 and CS2 core protein regions (Hayes and Melrose, 2020a). Some CS chains in neural aggrecan are replaced by HNK-1 trisaccharide which also attaches to the same core protein linkage tetrasaccharide as CS. Once the HNK-1 trisaccharide is assembled chain elongation ceases resulting in a reduction in CS chain density but introduces cell interactive properties. Neural aggrecan guides neural crest progenitor cell migration during embryonic neurogenesis and formation of the neural tube and notochord (Hayes and Melrose, 2020a). Preclinical spinal cord injury (SCI) and traumatic brain injury (TBI) animal model studies demonstrate that the enzymatic degradation of CS-PGs from gliotic scars using chondroitinase ABC improves neuronal functional recovery (Bradbury and Carter, 2011;Cheng et al., 2015;Muir et al., 2019). Endogenous degradation of the core protein of CS-PGs by ADAMTS-4 also improves neuronal functional recovery (Tauchi et al., 2012). While the CS-A and CS-C side chains of the lecticans inhibit neural repair, not all CS-PGs inhibit axonal re-growth (Mencio et al., 2021). PGs containing over-sulfated CS-B, and CS-E promote neurite outgrowth and functional recovery (Bovolenta and Fernaud-Espinosa, 2000). The EGF-like motif in the G3 domain of the lecticans has also been shown to regulate cell migration and tissue repair (Aguirre et al., 2007;Du et al., 2010). Overexpression of human EGFR in CNP (hEGFR) mice accelerates remyelination and functional recovery following focal demyelination. Progenitor cells overexpressing NG2 PG also improve re-myelination through EGFR mediated cell signaling (Keirstead and Blakemore, 1999;Aguirre et al., 2007). PNNs surrounding the soma and dendrites of a number of neuronal cell types are prevalent during neural development and maturation (Carulli and Verhaagen, 2021). A similar structure, the perinodal ECM surrounds the axonal nodes of Ranvier and appear after re-myelination, acting as a protective ion-diffusion barrier (Bekku and Oohashi, 2019;Fawcett et al., 2019). Perinodal structures in the Nodes of Ranvier also contain PNN components such as brevican and versican V2 (Bekku et al., 2009;Dours-Zimmermann et al., 2009).
of Alzheimer's disease (AD) (Morawski et al., 2012), cortical regions with abundant levels of ECM CS-PGs are less affected by degenerative features associated with the development of AD (Bruckner et al., 1999). PNNs also have important roles to play in Schizophrenia and Bipolar disorder (Berretta, 2012;Mauney et al., 2013). In unaffected individuals, the density of PNNs in the prefrontal cortex increases during pre-puberty and early adolescence. However, in patients with schizophrenia, a 70% reduction in PNN numbers in the prefrontal cortex has been observed (Mauney et al., 2013). The organization and function of PNNs is also disturbed in bipolar disorder (Gandal et al., 2018). Stem cells have been administered to promote recovery of normal PNN structure in an attempt to reverse these debilitating conditions (Forostyak et al., 2014). With an appreciation of PNNs and their important contributions to synaptic stability (Miyata et al., 2018), plasticity, memory and cognitive learning in normal brain tissues this has led to the identification of abnormalities in PNN assembly or expression of PNN components associated with particular neurodegerative conditions (Yamaguchi, 2000;Wen et al., 2018). Thus PNNs have become a therapeutic focus in the treatment of these conditions (Dityatev et al., , 2010(Dityatev et al., , 2021.

NEURAL PROTEOGLYCANS Aggrecan
In the CNS/PNS, aggrecan core protein contains KS, HNK-1 trisaccharide and CS side chains (Hayes and Melrose, 2020a) that convey unique tissue-specific functional properties ( Figure 3A). Aggrecan's ability to form macro-aggregates with HA provides water imbibing, space-filling and matrix stabilizing properties to the PNS/CNS ECM and in brain establishes ionic gradients and microcompartments important for the optimal activity of neural cell populations. The ability of the aggrecan core protein to assemble CS and KS chains at high density provides its well-known water-imbibing properties. Specific arrangements of GAG chains on aggrecan are functional determinant providing unique tissue contextdependent regulatory properties over neural cell populations. The aggrecan core protein KS and CS side chains and N-linked oligosaccharides all display neurite outgrowth-inhibitory activity (Hering et al., 2020). The cell mediatory properties of aggrecan's GAGs thus convey diverse regulatory roles in tissue development and in neuroprotective matrix stabilization of PNNs. Variation in the sulfation position and density on the CS side chains can influence morphogen and growth factor binding relevant to tissue development (Reichsman et al., 1996;Nandini and Sugahara, 2006;Nadanaka et al., 2008;Whalen et al., 2013;Mizumoto et al., 2015).

Neurocan
Neurocan has a widespread distribution in the CNS/PNS and is a component of PNNs (Schmidt et al., 2020) and regulates synaptic signaling (Sullivan et al., 2018). Neurocan ( Figure 3C) has roles in neurodegenerative disorders (Lin et al., 2021). β-amyloid increases neurocan expression in astrocytes through Sox9 influencing the development of AD. Mutations in the neurocan gene predispose to bipolar disorder and schizophrenia (Mühleisen et al., 2012;Raum et al., 2015). Neurocan regulates neural migration and axonal development in the cerebral cortex influencing the folding of the occipital and pre-frontal lobes and an increased probability of developing schizophrenia (Schultz et al., 2014).

OTHER NON-LECTICAN LARGE NEURAL PROTEOGLYCANS Phosphacan/Receptor Protein Tyrosine Phosphatase-Zeta (RPTP-ζ)
A cell membrane bound precursor form of phosphacan (RPTP-ζ) (Figures 4A,B) is processed by proteases to release a soluble PG ectodomain ( Figure 4C) called phosphacan (Chow et al., 2008), truncated and non-GAG substituted forms of phosphacan have also been described ( Figure 4D) with neurite outgrowth promoting activity (Fujikawa et al., 2017). This property is thus due to the core protein in some phosphacan species while neurite outgrowth activity may also be conveyed by GAG components, such as oversulfated CS-B and CS-E, in other phosphacan glycoforms (Dobbertin et al., 2003;Hikino et al., 2003). Phosphacan populations bearing KS and HNK-1 have also been described as well as the more common CS-glycanated form (Melrose, 2019b). Phosphacan promotes PNN formation (Eill et al., 2020). RPTP-ζphosphacan contain extracellular carbonic anhydrase (CAH) and fibronectin type III repeat domains, which foster protein-protein interactions (Milev et al., 1994;Lamprianou et al., 2011). A truncated 90 kDa phosphacan form is not a PG, but is substituted with the HNK-1 trisaccharide which facilitates interactive properties with a number of cell adhesion and ECM molecules . Phosphacan promotes neuron-glial interactions, neuronal differentiation, myelination, and axonal repair. The CAH carbonic anhydrase domain of phosphacan promotes protein-protein recognition, induces cell adhesion, neurite outgrowth of primary neurons, and differentiation of neuroblastoma cells (Adamsky et al., 2001); contactin is a phosphacan neuronal receptor that regulates neural development and axonal repair.

THE SMALL NEURAL PROTEOGLYCANS
Neuroglycan C Neuroglycan C is a part time  brain specific TM (Watanabe et al., 1995;Yasuda et al., 1998;Shuo et al., 2004) 150 kDa CS-PG ( Figure 4J) with a 120 kDa core protein that can also be shed by MMPs (Shuo et al., 2007), GAG-free forms of CSPG5 have also been described. Neuroglycan C, is a novel member of the neuregulin family (Kinugasa et al., 2004), interacting with pleiotropin (Nakanishi et al., 2010) producing neurite outgrowthpromoting activity mediated by phosphatidylinositol 3-kinase and protein kinase C (Nakanishi et al., 2006). CSPG5 forms peri-synaptic structures in the postnatal adult rat cortex (Jüttner et al., 2013). Impaired CSPG5 properties are evident in schizophrenia (So et al., 2010). Alternatively spliced forms have been identified in the human brain, recombinant CSPG5 induces phosphorylation of Erb2 and Erb3 and induces proliferation of neocorticol neurons (Kinugasa et al., 2004;Nakanishi et al., 2006). The neurite outgrowth promoting activity of neuroglycan C resides in its EGF and acidic amino acid domains (Nakanishi et al., 2006).

Syndecans
Syndecan transmembrane HS/CS-PGs (Figures 4E,F) modulate cell adhesion, cell-cell interactions and ligand-receptor interactions that regulate neural plasticity, promote neural growth and development (Couchman et al., 2015;Gopal et al., 2021). Sdc-3 and Sdc-4 are found throughout the nervous system and have roles in motor neuron development (Liu et al., 2020), Slit/Robo signaling and guidance of axonal development (Steigemann et al., 2004). Sdc3 is a co-receptor for Heparin-Binding Growth-Associated Molecule (HB-GAM)/midkine-induced neurite outgrowth in perinatal rat brain neurons. HB-GAM acts as a local, synaptic factor that promotes presynaptic and postsynaptic differentiation during neural development. Sdc3 also has roles in adult neuronal synaptic plasticity in the hippocampus in rat models following injury and regulates the neuronal internodal axonal ECM during re-myelination in growth, remodeling and repair (Steigemann et al., 2004). Sdc3 and Sdc4 promote functional recovery of neural tissues re-organizing sodium and potassium channels (Steigemann et al., 2004). Oligodendrocytes are sensitive to electro-stimulation, and this maintains their membrane polarization required for the promotion of axonal repair processes. Sdc1 is upregulated by neurons following TBI and SCI (Murakami et al., 2015). Sdc1 and Sdc3 knockdown in dorsal root ganglia (DRG) neurons induces short neurite extensions suggesting roles in nerve regeneration, synaptic formation and plasticity (Akita et al., 2004;Steigemann et al., 2004). Syndecans shed from the cell surface by MMPs, act as soluble growth factor co-receptors that regulate cell migration acting antagonistically with cell surface syndecans competing for FGF and VEGF binding  and interact with integrins potentially influencing cellular behavior, adhesion, spreading, migration, proliferation, tissue morphogenesis and pathogenetic tissue changes (Couchman et al., 2015).

Decorin
Decorin ( Figure 3E) regulates cellular survival, migratory, proliferative and angiogenic signaling and collagen fibril formation, sequesters TGF-β and antagonizes receptor tyrosine kinase family members, including EGFR and IGF-IR (Schönherr et al., 2005;Iozzo et al., 2011;Neill et al., 2012). MayDay, a ∼12 kDa N-terminal chemotactic factor, generated by macrophage-induced MMP-12 cleavage of decorin, recruits mesenchymal stem cells (MSCs) to damaged tissue regions in vitro and in vivo, promoting tissue repair (Dempsey et al., 2020). In situ hybridization (ISH) has localized decorin in areas of microvascular proliferation within gliomas and may be a therapeutic target in anti-angiogenic therapy (Patel et al., 2020) or approaches targeting TGF-β activity in tumors (Birch et al., 2020). Decorin protects neuronal tissue from the damaging effects of anti-oxidants and neuroinflammation following TBI by inactivation and has anti-tumor activity by inhibiting glioma cell migration (Yao et al., 2016). Decorin inhibits TGF-β activity, fibrous scar formation in neural tissues following trauma.

Biglycan
Biglycan ( Figure 3F) is synthesized by astrocytes (Koops et al., 1996) and immune cells (Mohan et al., 2010) and has neurotrophic activity, stimulates glial cell proliferation (Kikuchi et al., 2000) and neuronal cell survival (Koops et al., 1996). It is part of the proteome of the normal human retrobulbar optic nerve  and is massively upregulated around gliotic scars following trauma (Stichel et al., 1995). NF-κB upregulates biglycan, protecting human neuroblastoma cells from nitric oxide (NO)-induced cell death by inhibiting AMPK-mTOR mediated autophagy and intracellular reactive oxygen species (ROS) production from mitochondrial oxidative bursts , targeting Erk1/2 and p38 signaling pathways to prevent NO-induced neuronal cell apoptosis . Biglycan regulates neuroinflammation (Xie et al., 2020) through M1 microglial cell activation in the early stages of subarachnoid hemorrhage, targeting Erk1/2 and p38 signaling pathways . Biglycan binds to Notch-3 and accumulates in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) . Transcriptomic profiling of the hypothalamus and hippocampus, supports a central regulatory role for biglycan (bgn) in molecular pathways linking metabolic events with the immune response, and neuronal plasticity (Ying et al., 2018). Transcriptomic profiling of hypothalamus, hippocampus, and liver supports regulatory roles for Bgn in molecular pathways involved in metabolism, the immune response, and neuronal plasticity (Ying et al., 2018).

Epiphycan
The synaptic poles of inner hair cells of the cochlea have audiosensory properties and are surrounded by basket-like ECM structures with similar roles to the PNNs of neurons in the CNS (Sonntag et al., 2015). Epiphycan ( Figure 3G) and aggrecan are cochlear components and of the gel-filled tectorial membrane which detects auditory signals and transmits these to sensory hair cells (Melrose, 2019a). Epiphycan is expressed by cochlear supporting cells and is necessary for normal hearing. Epiphycan mRNA is abundantly expressed in the cochlea in the organ of Corti of neonatal and adult mice. The cochlea of epiphycan knockout (KO) mice display a normal morphology, however, the auditory brain-stem response is altered since epiphycan is necessary for normal auditory function (Hanada et al., 2017). These PNN like structures surround high function neuron types which respond to signals received from inner sensory hair cells, transducing audio signals into mechanical stimuli and receptormediated action potentials which are sent to spiral ganglion neurons (Sonntag et al., 2015). These neuron types operate at very high discharge rates and efficiently convey signals to the auditory brainstem for further processing. The hearing loss evident in cartilage matrix deficiency (CMD) mice is related to aggrecan deficiency in the cochlea (Melrose, 2019a).

Appican
Two variants of the related amyloid precursor-like protein 2 (APLP2) carry single CS-E side chains, which bind midkine and pleiotrophin (Thinakaran and Sisodia, 1994;Shioi et al., 1995;Thinakaran et al., 1995). Multiple splice variants of amyloid-beta precursor protein (APP) ( Figure 4I) and APLP2 arise from alternative splicing of three exons in APP and two exons in APLP2 (Kitaguchi et al., 1988;Ponte et al., 1988). The CS attachment site on APP/APLP2 is located adjacent to the membrane-spanning domain through deletion of 18 (APP) or 12 (APLP2) amino acids (Thinakaran et al., 1995). Splice variants also occur lacking CS side chains. APP and APLP are widely distributed in the CNS/PNS, APP is expressed by glial cells in the CNS/PNS.

Bikunin/ITI
Bikunin (inter-α-trypsin inhibitor light chain) is synthesized by neurons , occurring as a tissue form and small circulating PG containing a single CS chain ( Figure 3J). Bikunin displays anti-inflammatory, anti-protease, anti-microbial, antiviral properties and also functions as a growth factor (Fries and Blom, 2000;Lord et al., 2020). Bikunin is expressed in brain tissue (Takano et al., 1999;Kim et al., 2020) and accumulates in brain tumors. Bikunin CS chains contain embedded disulfated CS-D motifs (Lord et al., 2013(Lord et al., , 2020. A related Kunitz protease inhibitor, placental bikunin (hepatocyte growth factor activator inhibitor type-2) has been reported to inhibit glioblastoma tumor invasion (Hamasuna et al., 2001), however, this is a dissimilar protein to serum bikunin. Traumatic impact to the brain and spinal cord can release nuclear components such as histone H1 into the circulation or cerebrospinal fluid (CSF). Histone H1 has neuro-stimulatory effects and activates the innate immune response in the CNS mediated by microglial cells (Gilthorpe et al., 2013). This promotes neural cell survival, up-regulates major histocompatibility complex (MHC) class II antigen expression and is a powerful microglial chemoattractant. Release of histone H1 from the degenerative CNS drives a positive immune response (Gilthorpe et al., 2013) but can also be cytotoxic. Plasma immune tolerance induction (ITI) neutralizes the cytotoxic effects of histone H1, decreasing histone-induced platelet aggregation (Chaaban et al., 2015) through complexation of the histone with the negatively charged CS GAG chains of ITI (Chaaban et al., 2015). Hypoxic-ischemic encephalopathy predisposes infants to long-term cognitive decline impacting on life quality and healthcare resources (Chen et al., 2019). ITI regulates neonatal inflammation, decreases damage to brain tissues (Chen et al., 2019) and neuronal cell death, attenuates glial responses and leucocyte invasion with long-term beneficial effects in neonatal models of brain injury (Koehn et al., 2020).

Serglycin
Serglycin ( Figure 3H) is a small intracellular PG present in secretory granules of hemopoietic and endothelial cells (Kolset and Tveit, 2008) with regulatory properties over immune cells (Kolset and Pejler, 2011). It also promotes the development and aggressiveness of many tumor types including glioblastoma and is a glioblastoma biomarker (Roy et al., 2017;Manou et al., 2020). Suppression of serglycin in LN-18 shSRGN mutant cells results in retarded glioma proliferation, migration and invasive potential (Manou et al., 2020). Serglycin expression is elevated in astrocyteglioma co-cultures. Astrocytes promote glioblastoma growth and is a potential glioma therapeutic target (Mega et al., 2020).

Thrombomodulin (CD141)
Thrombomodulin ( Figure 4H) inhibits thrombin as part of the anticoagulant protein C-system in the endothelium, is anti-inflammatory and promotes barrier-stabilization. Thrombomodulin is a protective factor in the brain during ischemic stroke, enhancing vessel post-ischemic recovery in the blood brain barrier. Thrombin's physiological roles in the brain stabilize normal brain function in synaptic transmission and plasticity through direct or indirect activation of Protease-Activated Receptor-1 (PAR1) and has neuroprotective roles in neurological diseases (Krenzlin et al., 2016).

Cluster of Differentiation 44
Cluster of differentiation 44 (CD44), a major transmembrane glycoprotein HA receptor ( Figure 4K) in the CNS/PNS, has roles in cell division, migration, adhesion, and cell-cell and cell-ECM signaling (Dzwonek and Wilczynski, 2015). Alternatively spliced CD44v3 is a CS-PG, 20 isoforms of CD44 have been reported associated with several kinds of tumors. CD44 expression is highly dynamic and transitions between different isoforms during tumor development (Lah et al., 2020). In glioma, CD44 and integrins attach the cell to ECM forming focal adhesion complexes and generate traction forces that facilitate cell spreading, essential in the cell migratory machinery in glioma cell invasion (Mooney et al., 2016). In the normal brain, CD44 is a major HA receptor that interacts with osteopontin, collagens, and MMPs stabilisating and remodeling the CNS/PNS ECM (Dzwonek and Wilczynski, 2015). HA is highly interactive through CD44, conveying cell instructional cues, and ECM stabilization, hydration and spacefilling properties thus maintaining tissue compartmentalisation, ionic gradients and niches important in the metabolism of neural cell populations (Sherman et al., 2015;Peters and Sherman, 2020), including neural progenitor stem cell niches (Preston and Sherman, 2011).

Cluster of Differentiation 47
Cluster of differentiation 47 (CD47) (Figure 5A), originally named integrin-associated protein (IAP) is a receptor for thrombospondin-1 (TSP-1) regulates cellular migration, proliferation, and the survival of vascular cells, in innate and adaptive immune regulation (Barclay and Van den Berg, 2014;Murata et al., 2014). TSP-1 acts via CD47 to inhibit NO signaling in the vascular system supporting blood pressure by regulation limiting endothelial nitric oxide synthase (eNOS) activation and endothelial-dependent vasorelaxation. CD47 is a ligand for signal regulatory protein α (SIRPα), also known as SHPS-1/BIT/CD172a). The CD47-SIRPα signaling system is a cell-cell communication system Matlung et al., 2017;Weiskopf, 2017). CD47-SIRPα interactions have been termed an innate immune checkpoint in macrophages (Li et al., 2021). Blockade of anti-phagocytic CD47-SIRPα interactions using humanized antibodies to CD47 (Hu5F9-G4) has yielded promising results in preclinical studies of a number of human malignancies including pediatric brain tumors: medulloblastoma, atypical teratoid rhabdoid tumors, primitive neuroectodermal tumor, pediatric glioblastoma, and diffuse intrinsic pontine glioma (Gholamin et al., 2017) and accelerates the clearance of hematomas in experimental intraventricular hemorrhage (Ye et al., 2021). Thus by targeting the immunological checkpoint complex CD47-SIRPα, the development of glioblastoma can be inhibited, the function of phagocytic, dendritic and T-lymphocytes enhanced and the efficiency of tumor cell removal improved by innate and adaptive immune responses (Hutter et al., 2019;Hu et al., 2020;Kuo et al., 2020;Zhang et al., 2020).

Astrochondrin
Some specialized CNS proteins such as astrochondrin, a cell surface CS-PG of astrocytes carry L2/HNK-1 and L5 carbohydrate structures interactive with ECM components such as laminin and type IV collagen this may facilitate interaction of astrocyte foot processes with the brain microvasculature and meningeal membranes (Streit et al., 1993).

Photomedin
Photomedin is another brain-specific glycoprotein of the eye neuroepithelium that interacts with CS-E (Furutani et al., 2005). Photomedin is a member of the olfactomedin protein family and has regulatory roles in axonal growth and differentiation of sensory cilia in the neural epithelium.

CS-PGs REGULATION OF NEURONAL CELL SIGNALING
Neural cell populations including astrocytes, oligodendrocytes, neurons, endothelial cells and pericytes of the brain microvasculature and microglial cells synthesize a range of CS-PGs that interact with a variety of cell surface molecules and receptors (Figure 6). These modulate cellular processes that control CNS/PNS function and repair (Djerbal et al., 2017; Figure 7). CS-PGs contribute to the structural integrity and compartmentalisation of the brain ECM and have important organizational functional roles in brain regions (Dityatev et al., 2010). CS-PGs operate at multiple functional levels involving interactions with growth factors, receptors, adhesion molecules, neural guidance proteins and ECM proteins (Djerbal et al., 2017). Transmembrane CS-PGs are active during cell-cell crosstalk, they may also be secreted or released from the cell surface by proteases to act remotely from their cells of origin and may antagonize normal transmembrane PG interactions ( Figure 7F). CS-PGs are not uniformly distributed in the CNS/PNS but occur concentrated in neural growth cones and PNNs strategically positioned to control processes occurring at the cell-tissue interface (Shimbo et al., 2013;Sugitani et al., 2020). Growth cone receptor protein tyrosine phosphatases (RPTPs) bind with high affinity to CS-PGs, this controls axonal growth and provides guidance cues during regeneration, plasticity and neuronal development and in repair responses (Djerbal et al., 2017). CS-PGs attached to RPTP members can also exert repulsive guidance cues and inhibit neuritogenesis ( Figure 6A). Lectican PG family members (neurocan, brevican, versican, aggrecan) are diffusely distributed in the CNS ECM and are also components of the denser PNNs. The CS component of CS-PGs vary in composition with tissue development. During embryonic development CS-C is a predominant isoform while CS-A is more abundant in adult neural tissues (Figure 2). The sulphation patterns and charge density are important functional determinants of CS glycoforms. The more highly charged CS-E, B and D are components of PNN PGs, CS-A and CS-C are diffusely distributed in the ECM distant from PNN structures (Djerbal et al., 2017). The chemo-repellent semaphorin 3A (SEMA3A), a component of PNNs (Battistini and Tamagnone, 2016;Fard and Tamagnone, 2020), interacts with CS-E and B but not CS-D, thus such interactions are not purely mediated by charge; saccharide sequences in CS chains also determine the interactive properties of CS-PGs and their core proteins also contain ECM interactive modules. Selective binding of midkine (MK) and brain-derived neurotrophic factor (BDNF) to CS-E of some CS-PGs leads to neurite outgrowth. The Nogo receptors NgR1 and NgR3 bind to Nogo and inhibit neurite outgrowth. NgR1 and NgR3 also FIGURE 7 | Schematic representation of the structural organization of neural cell adhesion molecules that act as CS-PG receptors (A-C), and the neural NgR1 (D) and ephrin receptors (E) where binding of CS-PGs results in an inhibitory signal to the neuron. Ephrin acts as a receptor for Neuroglycan-C, while phosphacan binds to Contactin-1 (B) and NrCAM (C). LAR acts as a receptor for several CS-PGs. Oligodendrocyte-neuronal communication mediated by LAR-NrCAM (Condomitti and de Wit, 2018) or LAR-contactin-1 interactions  can be regulated by competitive binding of CS-PGs to these cell surface components (F).
bind specifically to CS-B, CS-D, and CS-E with high affinity, and this inhibits neurite outgrowth. Cell adhesion molecules are operative in cell-cell and cell-ECM interactions that regulate tissue integrity, cellular communication and cellular migration during CNS development and repair following trauma and are evident as pathological functional changes in neurological disorders. Neural cell adhesion molecule (NCAM) in particular, has specific roles in the promotion of neurite outgrowth of motor neurons that improves locomotor functional recovery following SCI. NCAM and neuroglia cell adhesion molecule (NgCAM) bind with high affinity to the CS-PG phosphacan, reducing neurite outgrowth and adhesion. Chondroitinase ABC moderately reduces phosphacan-NCAM binding showing this interaction is mainly mediated through phosphacan core protein interactions (Figure 7). Neurocan binding to NCAM and NgCAM also inhibits neurite outgrowth but unlike phosphacan, chondroitinase ABC abrogates this, showing that neurocan-NCAM interactions are mediated through CS. NCAM and NgCAM act as receptors for phosphacan and neurocan. Contactin-1 is a further GPI anchored cell adhesion molecule (CAM) that facilitates axonal growth and dendritic interactions that promote neurogenesis, CS-E binds contactin-1 with significant affinity and promotes neural growth. Thus, CS-PG binding to contactin-1 can modulate contactin-1 interactions that normally mediate cell-cell interaction ( Figure 7F). CS-PG5 (neuroglycan C) forms peri-synaptic matrix assemblies that regulate neuronal synaptic activity in the cerebral cortex of rats (Pintér et al., 2020).
The semaphorins are a family of guidance proteins of embryonic peripheral nerve projections and have roles in synaptogenesis and the maintenance of neural interconnections in adulthood thus maintaining cerebral homeostasis. SEMA3A is upregulated following CNS injury, causes growth cone collapse by signaling through synaptic neuropilin-1 (Nrp-1) and plexin receptors and is also known as collapsin-1. SEMA3A in PNNs can modulate synaptic morphology and function (Goshima et al., 2002;Mecollari et al., 2014;Battistini and Tamagnone, 2016;Alto and Terman, 2017;Fard and Tamagnone, 2020). SEMA3A interacts with CS-E with high affinity and neuropilin-1 (Nrp-1) in SEMA3A-Nrp-plexin signaling complexes to potently inhibit neural sprouting following SCI, and also inhibits neural growth factor (NGF) (Mecollari et al., 2014;Fard and Tamagnone, 2020) (Figure 6). Compared to SEMA3A, SEMA5A is less well studied, but also has important functional roles in CNS development and response to injury (Conrad et al., 2010). SEMA5A contains a cluster of thrombospondin (TSP) repeats which promote neural outgrowth. CS-PGs interact with this TSP repeat region producing a neuro-repulsive response whereas HS-PGs produce a neuro-attractive response. Thus, CS has important SEMA5A regulatory properties. A proteomic surface plasmon resonance and microarray study by Conrad et al. (2010), showed that interactions with neurotrophic factors was not confined to the highly charged CS-E glycoforms and that significant interactions also occurred between CS-A and Sema 3E, Sema 6B and ephrin A3 (Conrad et al., 2010).

CONCLUSION
This chapter has shown the impressive diversity in CS-PG form and function in the CNS/PNS. CS-PGs undertake many essential roles in neural tissues through the provision of a functional ECM for the many different cell populations resident in the CNS/PNS. The intricacies of some of the cell regulatory properties conveyed by CS-PGs have been illustrated, as has the complex interplay between distinct neural cell populations in the maintenance of CNS/PNS tissue function and homeostasis. Aberrations in the assembly of the ECM through processing defects in component CS-PGs can have serious functional consequences in brain tissues and can lead to neurodegenerative diseases. Such defects underline the fundamental importance of the ECM in normal tissue function and the potential of CS-PGs as promising therapeutic targets for future treatment of many of these neurodegenerative conditions.