Hyaluronan hydrates and compartmentalises the CNS/PNS extracellular matrix and provides niche environments conducive to the optimisation of neuronal activity

The central nervous system/peripheral nervous system (CNS/PNS) extracellular matrix is a dynamic and highly interactive space‐filling, cell‐supportive, matrix‐stabilising, hydrating entity that creates and maintains tissue compartments to facilitate regional ionic micro‐environments and micro‐gradients that promote optimal neural cellular activity. The CNS/PNS does not contain large supportive collagenous and elastic fibrillar networks but is dominated by a high glycosaminoglycan content, predominantly hyaluronan (HA) and collagen is restricted to the brain microvasculature, blood–brain barrier, neuromuscular junction and meninges dura, arachnoid and pia mater. Chondroitin sulphate‐rich proteoglycans (lecticans) interactive with HA have stabilising roles in perineuronal nets and contribute to neural plasticity, memory and cognitive processes. Hyaluronan also interacts with sialoproteoglycan associated with cones and rods (SPACRCAN) to stabilise the interphotoreceptor matrix and has protective properties that ensure photoreceptor viability and function is maintained. HA also regulates myelination/re‐myelination in neural networks. HA fragmentation has been observed in white matter injury, multiple sclerosis, and traumatic brain injury. HA fragments (2 × 105 Da) regulate oligodendrocyte precursor cell maturation, myelination/remyelination, and interact with TLR4 to initiate signalling cascades that mediate myelin basic protein transcription. HA and its fragments have regulatory roles over myelination which ensure high axonal neurotransduction rates are maintained in neural networks. Glioma is a particularly invasive brain tumour with extremely high mortality rates. HA, CD44 and RHAMM (receptor for HA‐mediated motility) HA receptors are highly expressed in this tumour. Conventional anti‐glioma drug treatments have been largely ineffective and surgical removal is normally not an option. CD44 and RHAMM glioma HA receptors can potentially be used to target gliomas with PEP‐1, a cell‐penetrating HA‐binding peptide. PEP‐1 can be conjugated to a therapeutic drug; such drug conjugates have successfully treated dense non‐operative tumours in other tissues, therefore similar applications warrant exploration as potential anti‐glioma treatments.


| INTRODUC TI ON
Proteoglycans (PGs) and hyaluronan (HA) make major contributions to the development and function of the brain. Chondroitin sulphate PGs (CSPGs) have instructive roles in axonal guidance and neurite extension Melrose et al., 2021;Mencio et al., 2021). Central and peripheral nervous system (CNS/ PNS) PGs are a diverse group of extracellular matrix (ECM) and cell membrane proteins that are decorated to variable degrees with all of the known glycosaminoglycan (GAG) classes. This huge GAG structural diversity equips CNS/PNS PGs with a broad range of cell directive properties through their ability to interact with a panoply of adhesive and structural glycoproteins, cellular receptors, neurotrophic factors, morphogens, growth factors, chemokines and cytokines. PGs are thus capable of influencing diverse cellular behaviours and processes, including cell proliferation, differentiation, adhesion and migration; and ECM synthesis and secretion, as occurs in tissue remodelling during development and in repair responses, including degenerative changes in pathological diseased tissues (Dyck & Karimi-Abdolrezaee, 2015;Mencio et al., 2021;Schwartz & Domowicz, 2018;Yang, 2020). Astrocytes, neurons and glial cell populations of the CNS/PNS all synthesise a diverse range of PGs that can be decorated with chondroitin sulphate (CS), keratan sulphate (KS), dermatan sulphate (DS), heparan sulphate (HS) and the human natural killer-1 (HNK-1) trisaccharide Lord et al., 2020;Melrose, 2019Melrose, , 2020Rushton et al., 2020). These GAGs have guidance roles during embryonic nerve development and formation of neural networks and neural microvasculature (Dyck & Karimi-Abdolrezaee, 2015;Mencio et al., 2021;Schwartz & Domowicz, 2018).

| HA NE T WORK FORMATI ON S TAB ILIS E S THE B R AIN ECM
PGs (lecticans) form specialised ECM structures known as perineuronal nets (PNNs), these are neuroprotective structures that provide synaptic plasticity (Testa et al., 2019); they contribute to the function of neuromuscular junction (NMJ) structures (Dani et al., 2012;Guarino et al., 2020;Rushton et al., 2020;Tezuka et al., 2014) of motor neurons that control muscular movement; and are integral components of the blood-brain barrier (BBB), where they maintain the integrity of this basement membrane structure (Nakamura et al., 2019;Steiner et al., 2014). Some PGs also interact with neuroregulatory receptors or are active at the neural cell surface, promoting the dimerisation and activation of neuroregulatory receptors that influence cell attachment and migration during neural development and CNS repair processes (Melrose, 2019). The correct assembly and/or processing of CNS/PNS PGs is critical for tissue functional properties: incorrect assembly or degradation of these PGs has been observed in a number of neurological disorders and may contribute to disease progression. Brain-enriched HAbinding brevican (BEHAB) is highly over-expressed in gliomas and contributes to the invasiveness and aggressiveness of these tumours Giamanco & Matthews, 2020;Matthews et al., 2000;Viapiano et al., 2008). NG2 PG/CSPG4 may have a role in the transformative stages of glioma development and may be a useful prognostic biomarker or vehicle for therapeutic drug delivery (Al-Mayhani et al., 2011).
Inter-alpha trypsin inhibitor (ITI) is a CS-PG ubiquitously expressed in brain tissues that co-localises with neurons and astrocytes in developing and adult human (Kim et al., 2020;Spasova et al., 2014) and rat brains (Chen et al., 2016). The direct interaction of HA and transferred ITI heavy chains plays a critical role in the organisation and stabilisation of the brain ECM. Administered ITI ameliorates brain injury, prevents cell death and improves behavioural outcomes after neonatal hypoxic ischaemia in rats Disdier et al., 2018;Lord et al., 2020). Since HA conveys critical functional aspects to neural tissues but is a soluble ECM component, effective means of immobilising HA in brain tissue is important for the maintenance of neural tissue function. Cross-linking of HA by transfer of ITI heavy chains is a potential means of immobilising and stabilising HA in the brain, cross-linking of HA is a protective mechanism in inflammation (Day & de la Motte, 2005), TSG-6 is a multifunctional protein with anti-inflammatory and tissue-protective properties that promote such HA cross-linking reactions (Day & Milner, 2019), networks. Glioma is a particularly invasive brain tumour with extremely high mortality rates. HA, CD44 and RHAMM (receptor for HA-mediated motility) HA receptors are highly expressed in this tumour. Conventional anti-glioma drug treatments have been largely ineffective and surgical removal is normally not an option. CD44 and RHAMM glioma HA receptors can potentially be used to target gliomas with PEP-1, a cell-penetrating HAbinding peptide. PEP-1 can be conjugated to a therapeutic drug; such drug conjugates have successfully treated dense non-operative tumours in other tissues, therefore similar applications warrant exploration as potential anti-glioma treatments.

K E Y W O R D S
CNS/PNS, extracellular matrix, glioma, HABPs, hyaluronan, SPACRCAN | 639 MELROSE furthermore, such enzymatically cross-linked HA hydrogels support the formation of 3D neural networks (Broguiere et al., 2016). Interalpha inhibitor proteins have been shown to ameliorate brain injury and improve behavioural characteristics in newborn and young rats exposed to hypoxic-ischaemic conditions .

| Hyaluronan is a major functional component of the CNS/PNS ECM
An unusual feature of the CNS/PNS ECM is that it is composed primarily of GAGs; for example, HA, CS, a major side chain component of PGs such as neurocan, brevican, versican, aggrecan and phosphacan, KS, HNK-1 trisaccharide and DS are also components of additional PG populations (Rauch et al., 2001). These interact with HA and structural and cell adhesive glycoproteins (e.g. tenascin-C, R; NCAM) forming network structures. Unlike weight-bearing connective tissue, the ECM of the CNS/PNS contains low levels of supportive fibrous proteins (e.g. collagens and elastin) (Burnside & Bradbury, 2014;Dityatev et al., 2010). Interestingly, GAG content varies considerably with age: HA, CS and HS levels all increase in rat brain tissue after birth reaching a peak at 7 days of age, thereafter GAG levels rapidly decline up to 30 days of age when GAG levels are within 10% of values in the adult brain. HA shows the most rapid decline after birth, decreasing by 50% in the first 3 days of life, attaining levels present in adult rat tissues (28% of peak HA values) by 18 days of age. Similar changes have been noted in the chicken brain. Furthermore, in 7-day-old rats, almost 90% of the HA in brain is extractable by water alone, compared to ~15% in adult animals reflecting HA interactions with matrix PGs and HA receptors that retain HA in adult brain tissues. Approximately 60% of the total GAG in brain tissues is HA (Figure 1). HA has important roles to play in the hydration and organisation of compartments and micro-gradients in the brain maintaining focal ionic balances.

| An appreciation of the central functional importance of HA in the CNS/PNS ECM
HA is a central component of the brain ECM with pivotal roles in neural development and plasticity. Brain HA exists both as a diffuse component soluble in saline and detergents and a condensed ECM in PG aggregate formations in PNNs which predominantly contain large-molecular-weight HA networked with lectican CS-PGs (Egorova et al., 2023). The brain ECM surrounds the space around and between neurons, glia and astrocytes and constitutes ~20% of the total brain volume. Compared to connective tissues, that are designed to withstand tension, compression or shear forces, the ECM of the CNS/PNS is unusual in that it does not contain appreciable levels of fibrous collagenous and elastic supportive networks (Burnside & Bradbury, 2014;Dityatev et al., 2010). HA is a major supportive component in the CNS/PNS with critical space-filling roles that provide tissue hydration, a matrix for cell attachment and properties conducive to cellular migration during CNS/PNS development. The immobilisation of HA in the CNS ECM is critical to the optimal functional properties of brain tissues; however, HA is a soluble polymer and it relies on essential interactions with PGs, HA receptors and HA interactive glycoproteins for its retention in the brain tissues ( Figure 1). HA undergoes depolymerisation during neuroinflammation, contributing to neurodegeneration, high-molecularweight HA is anti-inflammatory but low-molecular-weight HA and oligosaccharides have inflammatory properties inducing MMP synthesis and activation contributing to pathologic changes in tissues in neurodegenerative disorders of cognitive decline. Fragmented HA stimulates the expression of inflammatory genes by a variety of immune cells at the injury site and acts as an immune regulator (Jiang et al., 2011). HA binds to a number of cell surface proteins.
HA fragments signal through Toll-like receptor (TLR) 2, TLR4 and CD44 to stimulate genes in inflammatory cells. Remodelling of the brain ECM following injury in disorders such as perinatal hypoxicischaemic cerebral injury, multiple sclerosis and age-related vascular dementia results in depolymerisation of HA (Srivastava et al., 2020).
This inhibits myelin repair through the actions of specific-sized HA fragments which selectively block the maturation of late oligodendrocyte progenitors via an immune tolerance-like pathway that suppresses re-myelination (Srivastava et al., 2020). HA-binding protein involved in HA depolymerisation (HYBID), KIAA1199/CEMIP (cell migration-inducing and hyaluronan-binding protein), is a key player in HA depolymerisation in skin (Yoshida & Okada, 2019), arthritic synovial fibroblasts and brain (Yoshino et al., 2017(Yoshino et al., , 2018. HYBID knock-out (KO) mice display impaired spatial memory (Yoshino et al., 2018). HYBID, KIAA1199/CEMIP has roles in the initial stages of HA depolymerisation in skin. Mouse transmembrane protein-2 (mTMEM2) displays high structural similarity to HYBID and has been proposed to be a membrane-bound hyaluronidase; however, human TMEM2 does not degrade HA and does not act as a catalytic hyaluronidase but is a regulator of HA metabolism. Up-regulation of hTMEM2 expression by pro-inflammatory cytokines such as IL-1β decreases HYBID expression and increases hyaluronan synthase 2 (HAS2)-dependent HA production. Thus, hTMEM2 is a regulator of HA metabolism and not a hyaluronidase like in the mouse (Sato et al., 2023). Depolymerised HA and HA oligosaccharides stimulate the synthesis of MMPs by many cell types (Fuller et al., 2018;Hanabayashi et al., 2016;Ohno et al., 2006), which may result in focal tissue degeneration and generation of free radicals. Brain tissue is rich in phospholipids that are susceptible to peroxidation during neuroinflammation and by dysfunctional mitochondrial activity which releases oxygen, carbonate and dichloride free radicals capable of degrading HA (Al-Assaf et al., 2006;McNeil et al., 1985McNeil et al., , 1986).

| HA and glioma targeting
While HA has protective roles in brain development, it also has roles in the development of glioblastoma tumour masses. The high expression of the HA receptors CD44 and RHAMM by glioma cells could be potentially used to target these tumours with HA-conjugated drugs (Pibuel et al., 2021). HA is an extremely versatile carrier molecule amenable to conjugation with therapeutic agents using varied chemistries. HA has been conjugated with a number of cytotoxic drugs in hydrogel, micelle, nano-particle and liposome formulations for drug delivery with the amphiphilic properties of the HA adding to the versatility of the delivery process. The targeting of tumour cells using HA has been intensely investigated (Arpicco et al., 2014;Chen et al., 2014;Dosio et al., 2016;Edelman et al., 2017;Rao et al., 2016;Saravanakumar et al., 2014) and some particularly innovative systems have been developed (Shah et al., 2017;Zheng et al., 2016) offering improved delivery of compounds to solid tumours previously F I G U R E 1 Hyaluronan receptors, HA interactive proteins and proteoglycans with the BX7B HA-binding motif of RHAMM (a), cartilage link protein modules (b) and TLR-2 and TLR-4 (c). Schematic of the G1 globular HA-binding region of the lectican proteoglycan family with its immunoglobulin domain A and link B, B′ modules (d). Immunolocalisation of PNNs using monoclonal antibody 1B5 in the frontal mechanosensory cerebral cortex (e). Image (e). supplied by Dr AJ Hayes and Prof B. Caterson, Cardiff University, UK. BX7B, BX7B HAbinding motif (BX7B is a generic HA-binding synthetic peptide identified from HA-binding studies based on RHAMM); RHAMM, receptor for HA-mediated motility; Cdc 37, Hsp90 chaperone protein kinase-targeting subunit; HABP-1, HA-binding protein-1; IHABP4, intracellular HA-binding protein-4; SPACRCAN, Interphotoreceptor Matrix Proteoglycan-2; ITIHC3, inter-trypsin inhibitor heavy chain-3; LYVE-1, Lymphatic vessel endothelial hyaluronan receptor 1; HARE, HA receptor for endocytosis (Stabilin-2); CLEVER-1, vascular endothelial scavenger HA receptor (stabilin-1); BEHAB, brain-enriched HA-binding brevican fragment; TSG-6, Tumour necrosis factor-inducible gene 6 protein/TNF-stimulated gene 6 protein; Bral-1, Brain-specific HA-binding link protein; HAPLN-2, HA and proteoglycan link protein 2 (Bral-1); HABP2/PHBP, HA-binding protein-2/ HA-binding protein; GHAP, glial hyaluronic acid-binding protein; Versikine, Versican N-terminal HAbinding peptide; SHAP, serum-derived HA-associated protein (HC chain of ITI); TLR, Toll-like receptor.
Moreover, some of these probes can also be used to image the tumour mass and confirm delivery of the therapeutic compound, the size of the tumour mass and diminishment of the tumour size over time (Saravanakumar et al., 2014). Furthermore, the HA localised in the tumour mass could also potentially be used to target glioblastoma' using the HA-binding peptide PEP-1, which is a cell-penetrating peptide that has already found application in the transport of cargo proteins to target cells. PEP-1 has even been used to transport mitochondria into tumours in an attempt to control tumour growth (Chang et al., 2019). PEP-1 offers improved penetration of therapeutic drugs through the BBB and has already been used to target glioma cells Jiao et al., 2017;Wang et al., 2017).
Anti-angiogenic peptides have proved singularly ineffective in the treatment of glioblastoma.
HA is one of the major physiological barriers to the effective delivery of drugs to solid tumours and its targeting with the use of pharmaceutical agents has been used to decompress tumour blood vessels, which improves tumour perfusion and the efficacy of cytotoxic drugs (Papageorgis et al., 2017). Emerging therapeutic strategies that target the tumour microenvironment are being developed to improve vessel functionality and drug delivery (Voutouri et al., 2016). HA-CD44 interactions have crucial roles not only in malignancy but also in the resistance to cancer therapy (Kudo et al., 2017). Interference with HA-CD44 interactions by CD44targeting drugs is a strategy that has been developed using nanotechnology for cancer treatment delivering enhanced permeability and retention of therapeutic drugs (Skandalis et al., 2014). The cellpenetrating HA-binding peptide PEP-1 could further improve such delivery processes. Gliomas, a WHO grade IV astrocytoma, is the most common and deadliest form of brain cancer accounting for more than 50% of primary brain tumours. The ECM-penetrating capability of gliomas makes it impossible to treat these tumours surgically or to use radiotherapy. HA is a critical factor for glioma invasion and is also driven by up-regulation of MMP secretion, dissolution of ECM components and directed cell migration (Park et al., 2008).
Improved strategies for the treatment of gliomas are clearly required, and PEP-1 may represent a novel delivery system worth further evaluation (Cho et al., 2008;Jiao et al., 2017;Muñoz-Morris et al., 2007).

| The BX7B-HA-binding motif
Peptide ligands that bind specifically with high affinity to the recombinant HA-binding domain of RHAMM have been identified by screening 8-mer peptide libraries (Ziebell et al., 2001). The binding peptide is referred to as BX7B, where B represents any basic amino acid and X any non-acidic amino acid in the 8-mer peptide. Binding of BX7B peptide modules with RHAMM has been computer modelled using a three-dimensional NMR-based model of RHAMM. The human protein atlas shows Cdc 37 (cell division cycle protein-37) is widely expressed in human brain tissues and has roles in the stabilisation of Tau protein in microtubular structures and its phosphorylation (Jinwal et al., 2011). Cdc37 is a co-chaperone to Hsp 90 and forms a complex that controls the folding of a large number of protein kinases and thus has roles in intracellular signalling networks, some of these are linked to cancer development (Calderwood, 2015).
Cdc37 also binds HA and may have a role in the regulation of cell division. CD38 is an HA-binding protein ecto-enzyme of NAD glycohydrolase cell surface antigen (Nishina et al., 1994) that is strongly expressed in brain cells including neurons, astrocytes, as well as microglial cells, and is implicated in processes that lead to neurodegeneration and neuroinflammation (Guerreiro et al., 2020). Knockout of CD38 suppresses glial cell activation and neuroinflammation in a demyelination mouse model (Roboon et al., 2019). HA-binding protein 1 (HABP1) is identical to the splicing factor-associated protein p32 and the receptor of the globular head of the complement component gC1qR and has roles in tumorigenesis and cancer metastasis (Saha & Datta, 2018). HABP1, also known as p32, is a ubiquitous sialic acidsubstituted glycoprotein that functions in spermatogenesis and as a receptor for pro-inflammatory molecules. HABP1 not only localises to the Golgi apparatus but also has a widespread cytoplasmic distribution where it has roles in cellular replication (Saha & Datta, 2018). HABP4 (IHABP4) is a 57 kDa nuclear and cytoplasmic protein that interacts with several classes of cellular molecules, including proteins, RNA and HA, and is highly expressed in brain tissues with roles in the regulation of transcription and mRNA translation. Although capable of binding HA, this appears not to be the physiological target of IHABP4 (Huang et al., 2000). HABP4 is also known as Ki-1/57 which specifically interacts with the chromo-helicase-DNA-binding domain protein 3, a nuclear protein involved in chromatin remodelling and transcription regulation (Nery et al., 2004). The human protein atlas shows HABP4 is an intracellular protein localised to the cytoplasm and nuclear membrane. Besides binding HA HABP4 is an RNA-binding protein that plays a role in the regulation of transcription, pre-mRNA splicing and mRNA translation. IHABP4 has been proposed to act as a tumour suppressor in colorectal cancer (Melo-Hanchuk et al., 2020). HA regulates retinal development (Inoue et al., 2009) and is closely associated with versican, SPACR and SPACRCAN  expression where it may also have roles in the interphotoreceptor matrix of the mouse and rat retina and neural network development (Acharya et al., 1998(Acharya et al., , 2000Chen et al., 2003;Foletta et al., 2001;Salido & Ramamurthy, 2020).
SPARCAN is an HA-binding proteoglycan and SPACR is a related glycoprotein, both have important roles to play in interphotoreceptor matrix development and the attainment of vision. The heavy chain (HC3) of interα-trypsin inhibitor (ITI) contains a BX7B HA-binding motif (Jean et al., 2001); transfer of HC3 to HA forms a more condensed stable structure. Serum HA-binding protein (SHAP) is an ITIderived HA-binding protein found in serum (Zhao et al., 1995). ITI/ bikunin is a multifunctional, regulatory brain protease inhibitor PG (Chen et al., 2016;Mizushima et al., 1998) with unique HA crosslinking properties transferring ITI HCs to HA which are cross-linked by a trans-esterification process catalysed by TSG-6 (tumour necrosis factor-inducible gene 6 protein) which is also synthesised in the brain (Bertling et al., 2016;Coulson-Thomas et al., 2016). Bikunin/ ITI's properties are exemplified in its HA cross-linking roles which facilitate accumulation of HA around the cumulus-oocyte; this is an essential step in the reproductive process (Suzuki et al., 2004).
Matrix accumulation does not occur around the cumulus-oocyte in bikunin knock-out mice and these are infertile. HA cross-linking by HCs stabilises HA and along with interactions with PGs and HA receptors aids in the retention of HA in tissues (Milner et al., 2007;Rugg et al., 2005). Without this normal balance, tissues become susceptible to inflammation and tumour formation (Huth et al., 2015;Wakahara et al., 2005;Yagyu et al., 2006). ITI deficiency in the mouse is associated with anxiety-like behaviour and learning difficulties caused by alterations in brain ECM structure. HAPLN2, a brain-specific form of cartilage link protein, also termed Bral-1, has been suggested as a therapeutic target in the treatment of neurological disorders (Wang et al., 2019). HAPLN2 has roles in the maintenance of high conduction velocities in nerves (Bekku et al., 2010).
Toll-like receptors 2 and 4 (TLR2 and TLR4) are transmembrane pattern recognition receptors that remove pathogen-associated molecular patterns (PAMPs) from biological fluids including HA fragments.

| The link module HA-binding motif
The link module or PG tandem repeat is comprised of an immunoglobulin domain attached to two link modules (Figure 1b,d,e). The link module is a domain of approximately 100 amino acids stabilised by two disulphide bonds (Figure 1e). The 3D structure of the link module from human TSG-6, a 35 kDa protein comprised of a singlelink module attached to a CUB (complement C1r/C1s, Uegf, and Bmp1) domain, has been determined (Kohda et al., 1996). TSG-6 forms covalent complexes with the HCs of ITI, catalysing their transfer to HA in a cross-linking trans-esterification reaction that stabilises HA (Milner et al., 2007;Rugg et al., 2005). A number of HA receptors have been identified that use the tandem link module for binding to HA including CD44, lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), layilin and stabilin-1 and -2. CD44 is a major HA receptor with a widespread distribution in brain tissues. Glioma cells express CD44 and it promotes the pathogenesis of glioma (Gudbergsson et al., 2020;Mooney et al., 2016); CD44 is expressed by microglia/macrophages following ischaemic stroke (Sawada et al., 2020). HA and CD44 have regulatory roles in the stem cell niche in neural stem/progenitor cell proliferation, differentiation, maturation and progression to specific cell lineages (Preston & Sherman, 2011). CD44 is a major HA receptor (Aruffo et al., 1990) and is significantly up-regulated in inflammation and cancer. CD44 is highly expressed in malignant gliomas correlating with the aggressiveness of this tumour (Misra et al., 2015). CD44 is a complex signalling molecule regulating diverse cell processes in the developing and adult nervous systems through interactive roles with HA in normal tissue development (Peters & Sherman, 2020). CD-44 and HA regulate adult hippocampal neural stem cell quiescence and differentiation (Su et al., 2017). Layilin is a novel transmembrane HA receptor containing a C-type lectin domain. Layilin is expressed in brain and trigeminal ganglion that interacts with merlin and radixin (Bono et al., 2005).  Harris et al., 2007;Weigel, 2019). HARE is a multi-ligand endocytic scavenger receptor clearing HA, CS-A, C, D and E; heparin, DS, acetylated LDLs, col I and col III N-propeptides, advanced glycation end products and nucleic acid fragments while Stab2 clears apoptotic, gram-negative and -positive bacteria and lymphocytes (Weigel, 2019) from the circulatory and lymphatic systems (Harris & Cabral, 2019;Harris & Weigel, 2008). HARE/Stab2 are transmembrane glial phagocytic receptors, expressed by ependymal brain ventricle cells, macrophages and endothelial cells (Pandey & Weigel, 2014). Stab2 is transiently expressed at the cell surface but is mainly resident as an intracellular protein (Falkowski et al., 2003;Harris et al., 2007;Weigel, 2019).
Hyaluronan-binding protein 2 (HABP2) is also known as factor VII-activating protease and in humans is encoded by the HABP2 gene (Choi-Miura et al., 1996). HABP2 is a plasma serine protease that binds HA and has cell adhesive properties. It is synthesised as a single-chain protein that undergoes autoproteolysis to form a functional heterodimeric molecule containing three EGF, one Kringle and a serine protease domain (Römisch, 2002).

HBP2 is a potent activator of plasminogen activators including
pro-urokinase and has roles in coagulation, fibrinolysis and modulates vascular permeability through the generation of bradykinin (Römisch, 2002). Figure 1 shows the range of HA interactive molecules, these serve to immobilise HA in the brain ECM and regulate brain cell populations.

| Lectican HA-binding proteoglycans and their interactive modules
Members of the lectican family form macro-aggregate structures with HA stabilised by tenascin-R and link protein. These structures, termed perineuronal nets (PNNs), are laid down around neurons and have neurogenic and neuroprotective properties and roles in synaptic plasticity (Figure 1e). Tenascin-C is also found in developmental brain tissues where it acts as an endogenous activator of TLR4, regulates chemotaxis, phagocytosis and pro-inflammatory cytokine production in microglia and also has roles in glioma development (Brösicke & Faissner, 2015;Giblin & Midwood, 2015). While members of the lectican PG family all contain two link modules in their N-terminal G1 domains, which allow them to interact with HA, proteolytic fragments of some of the lecticans also share these HAbinding properties. BEHAB/brevican (brain-enriched hyaluronanbinding protein) is restricted to brain tissues, is first detected in the late embryonic period and its levels peak in the first two postnatal weeks. In the embryo, BEHAB is expressed at highest levels in mitotically active cells. BEHAB regulates cellular proliferation and differentiation and is over-expressed in gliomas, contributing to tumour invasiveness (Gary et al., 1998;Giamanco & Matthews, 2020;Nutt, Matthews, & Hockfield, 2001;Nutt, Zerillo, et al., 2001). Glial HA-binding protein (GHAP) is a 64 kDa N-terminal fragment (extending to Glu 405 ) of versican V0 or V2 containing the HA-binding G1 domain (Westling et al., 2004). A 70 kDa N-terminal fragment of versican V1 (extending to Glu 441 ) is now referred to as versikine (Foulcer et al., 2015;Nagyova et al., 2020). Aggrecanase-cleaved N-terminal fragments of versican regulate the regression of ECM after apoptosis in embryonic interdigital webs (McCulloch et al., 2009;Nandadasa et al., 2014).
Metastatin, isolated from bovine nasal cartilage as a complex between link protein (38 kDa) and an 85 kDa tryptic fragment of aggrecan G1 domain, blocks formation of tumour nodules in the lungs of mice inoculated with B16BL6 melanoma or Lewis lung carcinoma cells (Liu et al., 2001). Metastatin BH-P, a 42-mer peptide that contains three HA-binding motifs, potently inhibits the proliferation and colony-forming capability of B16 melanoma cells by activating caspase 3 and 8 pathways to selectively trigger apoptosis in melanoma cells (Béliveau et al., 2002;Falardeau et al., 2001;Gingras et al., 2001Gingras et al., , 2003Sauder et al., 2002). A further modulatory peptide based on the GA HA-binding domain of aggrecan is PEP-1 (Mummert et al., 2000). PEP-1 is a 12-mer (GAHWQFNALTVR) HA-binding peptide module that inhibits neutrophil homing to sites of inflammation and has potent HA-binding properties (Mummert et al., 2000). Besides its HA binding and ability to inhibit leucocyte trafficking, PEP-1 also has useful properties for cargo protein delivery into cells, across the BBB with the PEP-1 protein transduction domain able to deliver a number of therapeutic cargo proteins.
HAPLN-2 is the major link protein found in brain tissues where it acts as a molecular glue, linking the lectican PG family to HA in macro-aggregate structures in PNNs and acting as an organiser of the brain ECM and key facilitator of neuronal function and plasticity (Bekku et al., 2010;Oohashi et al., 2015). A deficiency of HAPLN-2 leads to abnormalities in the brain ECM and dysfunctional neuronal activity (Wang et al., 2019). The presence of HAPLN-2 in neurofibrillary tangles and insoluble amyloid-type protein aggregates contributes to the development of PD (Wang et al., 2016). Amyloid formation is linked to the development of diseases in the human body; macro-molecular amyloid aggregates disrupt normal tissue function (Bekku et al., 2010). Pathogenic amyloid aggregates occur when previously healthy proteins lose their normal physiological properties with the formation of insoluble fibrous protein aggregates in so-called disruptive plaque formations around cells that detrimentally affect normal cellular activity.
Amyloids have received notoriety for their disruptive properties in neurodegenerative disorders of cognitive decline such as AD, HD and PD. HAPLN-2 levels are low in the frontal lobes of Schizophrenia patients, further illustrating roles for HAPLN-2 and HA in brain physiology and pathology (Wang et al., 2019). Besides its roles in ECM organisation, HAPLN2 has roles in the maintenance of neuronal conductivity with deficient levels of HAPLN-2 leading to a significant reduction in nerve conduction velocity (Bekku et al., 2010). ECM PG networks have important roles to play in the electro-conductive properties of nervous tissues, abnormal organisation of these structures in PD, AD and schizophrenia contributes to the impaired functional neurological activity that characterises these disorders (Bekku et al., 2010).

| SPACRCAN stabilises the interphotoreceptor ECM
Photoreceptors project from the outer retinal surface into a specialised glycocalyx, the interphotoreceptor matrix, which contains HA and two specialised proteoglycans, SPACR (sialoprotein associated with cones and rods) and SPACRCAN (sialoproteoglycan associated with cones and rods). The interphotoreceptor matrix must be stable enough to provide functional attachment of the retina to the outer eye wall yet be porous enough to allow movement of metabolites between these tissues. SPACRCAN may participate in the maturation and maintenance of the light-sensitive photoreceptor outer segment. HA interacts with SPACR and SPACRCAN in the interphotoreceptor matrix of the retina and has roles in the spatial organisation of these structures and is essential for their survival through interactions with the retinal pigment epithelium (Olivier et al., 2022). HA has roles in retinal development and also interacts with versican as well as SPACR and SPARCAN during retinal development. The synthesis and degradation of HA are highly regulated in the retina and associated with retinal functional properties (Inoue et al., 2009).
SPACRCAN is a large HA-binding CS-PG of 300-400 kDa, which is reduced in size to 230 kDa by digestion with chondroitinase ABC (Chen et al., , 2004 (Acharya et al., 2000). SPACR is a 150 kDa sialic acid-rich glycoprotein which may or may not contain CS depending on species. Human SPACR does not contain CS, whereas mouse SPACR has three CS consensus attachment sites; this may be a specific modification that alters the functional properties of the fovea (Acharya et al., 1998). The expression of SPACRCAN and SPACR differs during embryonic eye development with SPACRCAN expression peaking in E16-18, whereas SPACR expression steadily increases from E15-E19 (Foletta et al., 2001;Inoue et al., 2006;Lee et al., 2000). This suggests SPACRCAN and SPACR have differing roles to play in eye development processes (Foletta et al., 2001).

| THE FUN C TI ONAL INTER AC TIVE PROPERTIE S OF HA ARE RELE VANT TO NEUR AL FUN C TION
While HA has a simple structure, consisting of the repeat disaccharide N-acetyl-glucosamine glycosidically linked to D-glucuronic acid, this simplicity belies the highly interactive properties of this polymer with PGs, HA receptors and HA interactive glycoproteins (Figure 1), which create massive space-filling macro-aggregates and cell interactive structures. The high solvation volume of these structures is critical to brain volume, maintenance of cellular organisation and compartmentalisation that is essential for the homeostatic balance of ionic species and other metabolites in distinct cellular compartments of the brain. HA deficiency caused by Has3 knockout results in seizures in mice and HA synthase-3 knockout mice display an epileptic phenotype (Arranz et al., 2014;Perkins et al., 2017). The high-lipid and long-chain fatty acid content in brain tissues acts as an energy store. Phospholipids are also structural and functional components of the plasma membranes that anchor lipoproteins in CNS/PNS cells as well as provide the insulating properties of the myelin sheath that is essential for the maintenance of high-conduction velocities across nerves.
The extensive network of blood vessels in brain tissues is important to brain function, providing the high nutritional demands of the CNS/PNS. Although the brain only constitutes ~2% of total body weight, it utilises ~20% of the total oxygen and ~50% of the total glucose consumption of the human body. The maintenance of membrane potentials, through the action of Na + /K + voltagegated ion channels and ATPase, is essential for the transport of ions across membranes. These create and maintain ionic balances in brain compartments, thus the interactive properties of neurons are a major energy-consumptive process. The physical and metabolic activities of the neuron and astrocyte are intimately linked and essential for optimal neuronal functioning. The CNS/ PNS ECM facilitates cross-talk among glia, ECM and neurons (Song & Dityatev, 2018). Perineuronal nets formed from HA and members of the lectican PG family (Yamaguchi, 2000) are neuroprotective structures promoting synaptic plasticity and homeostasis (Dityatev et al., 2010) (Figure 2). CD44, a major HA receptor, acts as a synaptic cell-ECM interactive adhesion molecule that regulates structural and functional plasticity (Roszkowska et al., 2016) and has roles in the retention of spatial memory and sensorimotor function (Raber et al., 2014). The HA-based pericellular matrices assembled by glia and their sensory signals feed back to the cell following trauma, resulting in proteolytic remodelling of brain tissue. Cross-talk between glia and neuron cell populations co-ordinates brain and spinal cord repair processes following trauma (Song & Dityatev, 2018).

| Lecticans and PNNs
Lectican CS proteoglycans form macromolecular networks with HA stabilised by tenascin-R and brain-specific link protein (Bral2) called perineuronal nets (PNNs) (Aspberg et al., 1997;Miao et al., 2014;Yamaguchi, 2000). These protect neurons from mechanical and oxidative damage and provide synaptic plasticity making contributions to cognitive learning processes and memory.
Disruption in PNNs has been noted in neurological disorders including epilepsy caused by impaired synaptic signalling (McRae & Porter, 2012). Aggrecan is a major component of PNNs where its dense CS chains provide strength to the dense ECM surrounding PNNs, however, versican, brevican and neurocan have also been observed in these structures ( Table 1). PNN GAGs have roles in cognitive learning and memory (Duncan et al., 2019;Yang et al., 2021). GAGs are key functional components of PNNs and facilitate neuronal communication and neural plasticity (Richter et al., 2018;van 't Spijker & Kwok, 2017).
While CS, a major component of PNNs, is generally considered to provide inhibitory cues in glial scars through lectican CS-PG deposition limiting functional neuronal recovery following trauma, PNNs have been proposed to have roles that actively promote neural regeneration (Carulli et al., 2016;Kwok et al., 2011;Sorg et al., 2016;Yang et al., 2014). The sulphation of GAGs is a key functional determinant in PNNs, changes in PG sulphation with ageing may render these structures more inhibitory Foscarin et al., 2017) and may be as a result of changes in the expression of sulphotransferases responsible for GAG sulphation (Properzi et al., 2008). Manipulation of PNNs has been proposed to be capable of altering selective memories (Poli et al., 2023;Romberg et al., 2013). PNNs have also been proposed to modulate visual inputs to the brain (Faini et al., 2018).

F I G U R E 2
Schematic depiction of the structural organisation of SPACRCAN (sialoproteoglycan associated with cones and rods) as proposed by Hollyfield (2001) and Hollyfield et al. (2002) showing the central sialic acid-rich mucin domain and four CS chains plus the proposed RHAMM-like HA-binding regions and epidermal growth factor (EGF) domain and a region of hydrophobic amino acids. A few N-linked oligosaccharides along the core protein are also shown. The CS chain glucuronic acid-N-acetylgalactosamine components of the repeat disaccharides are indicated using SFNG icons for GAG components, and the sulphation of the N-acetyl galactosamine components is also shown.

| Stabilisation of HA in tissues by transfer of ITI H chains
ITI has the ability to transfer its heavy chains to HA in tissues where the heavy chains cross-link HA by a trans-esterification reaction catalysed by the enzyme TSG-6 (TNFα-stimulated gene/protein-6). This produces a denser form of HA aiding in its immobilisation and the stabilisation and protection of tissues (Oh et al., 2010). This may be particularly relevant to brain tissue which is one of the softest tissues in the human body. TSG-6 is constitutively expressed in the brain and spinal cord, up-regulated in response to inflammation and has antiinflammatory and immunomodulatory tissue-protective functions in neurodegenerative diseases (Day & Milner, 2019;La Russa et al., 2023).
Administered ITI ameliorates brain injury, prevents cell death and improves behavioural outcomes after neonatal hypoxic ischaemia in rats Koehn et al., 2022;McCullough et al., 2021;Schuffels et al., 2020). ITI is ubiquitously expressed in brain tissues, and co-localises with neurons and astrocytes in the developing and adult human (Kim et al., 2020) and rat brain (Chen et al., 2016). The direct interaction of HA and ITI H chains plays a critical role in the organisation and stabilisation of the brain ECM.

| CON CLUS IONS
The ECM of the CNS/PNS is a truly remarkable supportive and dynamic interactive structure containing a biodiverse collection of molecules that collectively regulate cellular behaviour and essential neural physiological processes. The CNS/PNS ECM is unique in that it does not utilise collagenous or elastic fibrillar networks or TA B L E 1 HA interactive proteoglycans of the peripheral and central nervous systems.
sheet-like structures to provide cellular support and tissue function as occurs in other weight-bearing, tension-and shear-resisting tissues.
The ECM of the CNS/PNS uniquely contains a high GAG content with HA, representing at least half of the total GAG present within the ECM and this provides matrix stabilisation. HA is a soluble GAG and displays sophisticated cellular interactions with many receptors and PGs, this ensures it is retained in neural tissues. This is important not only for the hydration of the CNS/PNS, which is a compliant tissue and one of the softest in the human body, but also for the functional status of resident neuron populations. Animal models show that a reduction in HA levels in brain tissues results in epilepsy, frequent convulsive episodes and alterations in social behaviour, thus it is important that optimal levels of HA should be maintained in the brain. The space-filling properties of HA ensure that the intricate compartmentalisation of the CNS is maintained and that ionic microenvironments are controlled to ensure that the polarisation dynamics of neural membranes are upheld for effective regulation of neural activation and neurotransduction. HA is thus a particularly important functional component of the CNS/PNS from the earliest embryonic developmental stages into maturity. HA also has essential roles in the interphotoreceptor matrix and regulates the spatial organisation and function of the photoreceptors involved in phototransduction and neurotransduction in the retinal neural network. Such photoreceptorneural signals supply ~60% of all sensory inputs to the brain which is in standing with sight being the primary human sense. Glioma is a particularly invasive tumour in the brain with extremely high mortality rates. HA is highly expressed in these tumours, which broadly express CD44 and RHAMM HA receptors. Conventional drug treatments have been largely ineffective against this tumour and surgical removal is normally not possible. Thus, a more effective treatment for gliomas is required. The CD44 and RHAMM HA receptors of gliomas can potentially be used to target them with PEP-1, a cell-penetrating HA-binding peptide which can be conjugated with a therapeutic drug.
PEP-1 drug conjugates have already been used successfully to treat dense non-operative tumours, thus similar applications warrant development for the treatment of glioma and are eagerly anticipated.

AUTH O R CO NTR I B UTI O N S
JM conceived the study, wrote the original draft and subsequent revisions.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The author has no conflicts to report.

PEER R E V I E W
The peer review history for this article is available at https://www. webof scien ce.com/api/gatew ay/wos/peer-revie w/10.1111/jnc.15915.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data is available in the cited bibliography

JM has received consultancy fees from Arthropharm and Sylvan
Pharmaceutical Companies Ltd.