Chondroitin sulphate proteoglycans: Extracellular matrix proteins that regulate immunity of the central nervous system

Autoimmunity Reviews 10 (2011) 766–772

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Autoimmunity Reviews j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a u t r ev

Review

Chondroitin sulphate proteoglycans: Extracellular matrix proteins that regulate immunity of the central nervous system Sarah Haylock-Jacobs, Michael B. Keough, Lorraine Lau, V. Wee Yong ⁎ Hotchkiss Brain Institute and the Department of Clinical Neurosciences, University of Calgary, Calgary, Alberta Canada

a r t i c l e

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Article history: Received 22 May 2011 Accepted 24 May 2011 Available online 2 June 2011 Keywords: Proteoglycan Multiple sclerosis Spinal cord injury Arthritis Extracellular matrix Repair Metalloproteinases Chondroitin sulphate

a b s t r a c t The extracellular matrix (ECM) is a complex network of scaffolding molecules that also plays an important role in cell signalling, migration and tissue structure. In the central nervous system (CNS), the ECM is integral to the efficient development/guidance and survival of neurons and axons. However, changes in distribution of the ECM in the CNS may significantly enhance pathology in CNS disease or following injury. One group of ECM proteins that is important for CNS homeostasis is the chondroitin sulphate proteoglycans (CSPGs). Up-regulation of these molecules has been demonstrated to be both desirable and detrimental following CNS injury. Taking cues from arthritis, where there is a strong anti-CSPG immune response, there is evidence that suggests that CSPGs may influence immunity during CNS pathological conditions. This review focuses on the role of CSPGs in CNS pathologies as well as in immunity, both from a viewpoint of how they may inhibit repair and exacerbate damage in the CNS, and how they are involved in activation and function of peripheral immune cells, particularly in multiple sclerosis. Lastly, we address how CSPGs may be manipulated to improve disease outcomes. © 2011 Elsevier B.V. All rights reserved.

Contents 1. 2.

The extracellular matrix: the glue that holds the CNS together . . . . . . . . . . . CSPG biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Methods of altering CSPG function . . . . . . . . . . . . . . . . . . . . . 3. Changes in CSPG distribution following injury or during disease; friend or foe? . . . 3.1. Spinal cord injury and the disadvantages of CSPG accumulation . . . . . . . 3.2. CSPG breakdown is detrimental in arthritis . . . . . . . . . . . . . . . . . 3.3. Multiple sclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. The benefits of CSPG expression in pathology . . . . . . . . . . . . . . . . 4. A co-conspirator? CSPGs and their direct/indirect interaction with peripheral immune 5. Looking to the future: manipulation of the CNS ECM for therapeutic benefit? . . . . 6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Take-home messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. The extracellular matrix: the glue that holds the CNS together The central nervous system (CNS) is a specialised cellular system that provides the body with the signals vital for life. Neurons are

⁎ Corresponding author at: University of Calgary, 3330 Hospital Drive, Calgary, Alberta, Canada T2N4N1. Tel.: + 1 11 403 220 3544; fax: + 1 11 403 210 8840. E-mail address: [email protected] (V.W. Yong). 1568-9972/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.autrev.2011.05.019

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supported by a cellular network that includes astrocytes, microglia and oligodendrocytes which function, respectively, to maintain synapses and blood brain barrier integrity, perform immune surveillance, and ensheath axons with myelin. Exterior to and between these cells is the extracellular matrix (ECM) which constitutes approximately 20% of total brain volume [1]. The ECM is integral for the structure and organisation of the CNS as well as for binding and presenting proteins such as cytokines, chemokines and growth factors to cells [2–6]. The ECM is therefore not only critical for the development and daily

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maintenance of the CNS, but it is also involved in the initiation and progression of CNS pathology. The major components of the CNS ECM include hyaluronan, tenascins, laminins, collagen, fibronectin, link proteins and proteoglycans such as the chondroitin sulphate-, dermatan sulphate- and heparan sulphate-proteoglycans. These components form a specialised mesh network that differs depending on the specific location in the CNS. For example, the basement membranes that are associated with blood vessels and help support the blood–brain barrier are rich in collagen, fibronectin and laminins [7] whilst the equally dense perineuronal nets around the soma of some neurons, and which are integral for maintaining synaptic plasticity [8], contain hyaluronan, tenascin-R, link proteins, and high levels of chondroitin sulphate proteoglycans (CSPGs) [9,10]. The neural interstitial matrix, which is diffusely dispersed between cellular structures and is rich in CSPGs and hyaluronan, contains most of the ECM components that are found in the CNS [11]. While the ECM is integral for everyday function of the mature CNS, alterations in ECM component expression often occur following CNS pathology such as gliomas, spinal cord injury, and inflammatory diseases such as multiple sclerosis (MS) [12–18]. Up-regulation of several proteins, particularly CSPGs, results in the generation of a ‘barrier’ to neuroregeneration [12–16]. In this review we focus on the role that CSPGs play in immunity of the diseased/injured CNS, particularly the putative autoimmune condition of the CNS, MS. We also take guides from an autoimmune condition in the periphery, arthritis, on the possible impact that CSPGs may have on cells of the immune system once they reach the CNS.

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membrane bound (on the surface of oligodendrocyte precursor cells (OPCs) in the CNS [27]), whereas the other proteoglycans are generally secreted into the ECM [24–26]. Each of the core proteins has different lengths and numbers of GAG chains attached, and the GAG chains themselves can have varying chondroitin sulphate sulphation patterns (Fig. 1B). In some cases, CSPG core proteins can bind hyaluronan and/or tenascin proteins [10,28], therefore aiding in the organisation of the ECM. Furthermore, CSPG core proteins can also be covalently bound to not only chondroitin sulphate GAG chains, but also dermatan sulphate GAG chains thereby increasing functional diversity within the proteoglycan family [29]. In the adult CNS, all of the major cell types (such as astrocytes, neurons, oligodendrocytes and microglia) are thought to be capable of making CSPGs and aiding in their arrangement in the ECM, particularly in perineuronal nets [30]. Whilst it is clear that CSPGs are critical for guidance of migrating cells within the developing CNS, a basal level of CSPG production is necessary in adulthood to maintain ECM structures in order to afford synapse stabilisation and plasticity as well as correct neural network structure and prevent improper organisation and sprouting of axons [30]. However, CSPG over-production in the CNS can be induced upon inflammatory stimulation, such as that which occurs following spinal cord injury and during MS [12,13,15–17,31] (see Sections 3.1 and 3.3). Astrocytes are thought to be primarily responsible for up-regulation of CSPGs in pathological environments following exposure to cytokines such as IL-1β, TGF-β and EGF [32].

2.1. Methods of altering CSPG function 2. CSPG biology

B

CH2OH O

O

OH

NHAc

CH2OSO3O

CH2OSO3-

COOH O HO

O

O-

OSO3-

NHAc

Phosphacan

Brevican

Neurocan

Aggrecan

Versican V3

Versican V2

Versican V1

Versican V0

O3-SO

COOH O HO

O NHAc

OH

Chondroitin sulfate E

Chondroitin sulfate D

NG2

OH

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O

OH

HO

O NHAc

Chondroitin sulfate A

COOH O

OH

O-

HO

O

O-

C-terminal

CH2OSO3 -

COOH O

O3-SO

O-

Core protein

A

N-terminal

The molecular characteristics of CSPGs have been extensively reviewed elsewhere [10,13,18–23]. In brief, CSPGs consist of a large protein core which is covalently attached to glycosaminoglycan (GAG) chains via a linking region (Fig. 1). The GAG chains are made up of repeating chondroitin sulphate (CS) disaccharide subunits linked to form varying lengths and are thought to mediate many of the binding interactions between CSPGs and other proteins. There are several submembers of CSPGs as determined by their protein core including aggrecan, brevican, versican, neurocan, NG2, and phosphacan (Fig. 1A) [24–26]. NG2 consists of a transmembrane region and can therefore be extracellular or

Several methods have been used to reduce CSPG expression or to degrade existing CSPGs in experimentally induced disease or injury (Table 1). Firstly, synthesis of CSPGs can be reduced by inhibitors that affect the GAG side chains. For example, fluorinated glucosamine analogues prevent the synthesis of chondroitin/heparan sulphate side chains by inhibiting an enzyme important for the elongation of polysaccharide chains [33]. Alternatively, GAG chains are built off of a characteristic xylose sugar in the linker region of the proteoglycan [34] (Fig. 1C), and the use of small xylose-conjugated organic molecules, such as 4-Methylumbelliferyl-beta-D-xylopyranoside (xyloside) reduces GAG synthesis machinery from attaching GAG chains to the CSPG protein core [35,36]. In turn, either of these strategies will reduce the amount of complete CSPGs released into the media [35].

C Core protein

Chondroitin sulfate GalNAC

Glucuronic acid

Galactose

Galactose

Xylose

Serine

Versicans Lecticans

n

Linkage region

Core protein

Fig. 1. Schematic diagram of CSPG family members, chondroitin sulphate subunits and the core-protein-GAG linking region. (A) The CSPG family consists of Aggrecan, Versican, Brevican, Neurocan (also known as Lecticans), Phosphacan, and NG2 (which can either be membrane-bound or extracellular). The CSPG core proteins generally contain an amino terminal which binds Hyaluronan and a carboxy terminal which binds tenascin proteins (Adapted from [24–26]). (B) The GAG chains consist of repeating chondroitin sulphate units that are designated CS-A, CS-C, CS-D or CS-E depending on the position of their sulphation (Adapted from [25]). (C) The GAG chains are attached to the core protein via a linking region (Adapted from [26,85]).

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Table 1 Methods of altering CSPG synthesis and function. Agent

Method of action

Result

References

4-fluoro-glucosamine

Inhibits CS/HS synthesis

[33]

p-nitrophenyl-beta-D-xyloside

Inhibits GAG chain attachment

4-methyl-umbelliferyl-betaD-xylopyranoside Chondroitinase ABC Metalloproteinases IL-1α

Inhibits GAG chain attachment

Reduction of GAG chain attachment to CSPG core proteins and thereby reduced core protein secretion to the ECM. Reduction of GAG chain attachment to CSPG core proteins and thereby reduced core protein secretion to the ECM. Reduction of GAG chain attachment to CSPG core proteins and thereby reduced core protein secretion to the ECM. Function of ECM CSPG is reduced. Clearance of CSPGs Cleavage of ECM CSPGs, particularly aggrecan.

Cleaves GAG chains from ECM CSPG Degrades core protein Increases ADAMTS4 expression

[35,86] [35,86] [37,38] [42–45] [42]

Table 2 The negative and beneficial roles of CSPGs in CNS trauma, MS, arthritis and immunity. Negative roles of CSPGs CNS trauma

Multiple sclerosis Arthritis

Immunity

Highly expressed in glial scar. Block axonal regeneration. Increase collapse of growth cones. Present at edges of MS lesions. CS GAGs ↑ Th1 and Th17 differentiation and worsen EAE. Breakdown of Aggrecan leads to irreversible ECM loss and chronic disability. Breakdown is mediated by ADAMTS proteins. Anti-aggrecan cell-mediated immune response. CSPGs bind chemokines (homeostatic and pro-inflammatory). CSPGs can bind to and activate CD44. CSPGs can interact with cell trafficking molecules (L- and P-selectin).

[16,31] [12–16] [51,52] [17] [64,65] [20,56–58] [61,62] [59,60] [2,5] [29,39] [29]

CSPGs can regulate microglia and enhance a regulatory phenotype at early time points post spinal cord injury CS disaccharides ↓ T cell migration and EAE pathogenesis.

[39,68] [64,65]

Beneficial roles of CSPGs CNS Trauma Multiple sclerosis

The function of CSPGs can also be altered after they have been excreted into the ECM. A direct approach is the use of chondroitinase ABC, an enzyme derived from Proteus vulgaris. Chondroitinase ABC acts by cleaving the chondroitin sulphate disaccharides, essentially leaving the core protein intact but without GAG chains attached [37,38]. As much of the CSPG function occurs via interactions between GAG chains and receptors like PTPσ and CD44 (see Sections 3.1 and 4) [29,39,40], chondroitinase ABC treatment resulting in GAG chain removal would therefore prevent these interactions [41]. CSPGs can be degraded from the extracellular environment by upregulation of the proteases involved in regular CSPG turnover. For example, the proinflammatory cytokine IL-1α up-regulates ADAMTS (‘A Disintegrin and Metalloproteinase with ThromboSpondin Motifs’)-4, a protein that normally degrades tissue aggrecan (see Section 3.2) [42]. Chronic peripheral inflammatory conditions can lead to tissue damage accounted for by sustained CSPG degradation, as occurs in arthritis. Finally, CSPGs can also be degraded by another class of proteases, the matrix metalloproteinases (MMPs), which can remove both the core protein and the GAG chains [43–45]. The activity on CSPGs may help account for the widespread detrimental and beneficial aspects of MMPs within the CNS [46–48]. While chondroitinase ABC and xyloside are the most common methods used to degrade CSPGs or inhibit CSPG biosynthesis, using small molecule inhibitors, siRNA, or employing methods to upregulate proteins such as the ADAMTSs, have the potential to reduce the CSPG burden during injury or disease [49]. 3. Changes in CSPG distribution following injury or during disease; friend or foe? In recent years there has been a significant increase in the amount of literature focussing on both the positive and negative aspects of CSPG expression following injury and during disease (Table 2). It is

well known that CSPGs are important for three-dimensional structure of tissues, particularly in the joints, and neuronal guidance in development and plasticity of neural synapses in adults. Despite this, increased expression of CSPGs can be a significant disadvantage following CNS injury, as described in the section below on spinal cord injury. However, there may be particular benefits to the CSPGs that accumulate during CNS injury, although this is gleaned from the literature on arthritis and in a single study following early spinal cord injury (see below). How CSPGs in the CNS affect MS is less well understood. The following section summarises the diverse roles of these proteins in several different pathological situations. 3.1. Spinal cord injury and the disadvantages of CSPG accumulation It has been widely reported that damage to the CNS results in the formation of CSPG-rich glial scar tissue that is primarily formed by reactive astrocytes [16,31]. The accumulated CSPGs are thought to be the main contributor of the failure of axonal regeneration through the damaged area [12,13,15,16,50]. For example, regenerating corticospinal tract axons that enter a barrier of CSPGs in the injured spinal cord fail to penetrate that barrier, effectively preventing further regrowth of axons [37]. In vitro, CSPGs have been shown to reduce neurite outgrowth through inducing the collapse of growth cones via the Rho/ROCK pathway [51,52]. In support of the non-permissive nature of CSPGs, the local treatment with chondroitinase ABC immediately following injury in vivo improves axon regeneration and functional recovery [37,53,54]. Thus, CSPGs are an important inhibitory contributor to the glial scar that arises following CNS injury. Recently, it has been reported that CSPGs function through the protein tyrosine phosphatase-sigma (PTPσ) receptor [40,55]. It is through this interaction with PTPσ that CSPGs are capable of inhibiting neurite outgrowth through CSPG gradients in vitro and spinal cord injury sites in vivo. Despite this, whilst PTPσ −/− mice

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displayed neurite outgrowth that extended further through the lesion in the injured spinal cord, damage to the area was still significant, indicating that other inhibitory proteins in the area are still sufficient to disallow complete repair and/or CSPGs can possibly act through receptors other than PTPσ (or through other PTP receptor isoforms) to inhibit axonal regeneration [40]. 3.2. CSPG breakdown is detrimental in arthritis While removal of the up-regulated CSPGs in the CNS would likely be beneficial following CNS injury, maintenance of ECM integrity in joints is critical. In normal joints, the highly-expressed CSPG aggrecan interacts with hyaluronan and links proteins to form large aggregates which, when the negatively charged GAG chains interact with water molecules, add to the compressible and elastic properties of the joint cartilage that are required for fluid movement [20,56]. Shortfalls in maintenance/repair and immunological onslaughts to this area are common in several different types of arthritis such as rheumatoid and osteoarthritis. In these conditions aggrecan core protein is degraded, resulting in irreversible ECM loss and chronic disability [20,56–58]. Studies in proteoglycan-induced arthritis have demonstrated that this disease appears to be primarily T-cell driven, but, due to the fact that there are very few T cells found in the synovial tissue of joints, it is thought that the T cells are primarily driving an auto-antibody response and contributing to the inflammatory cytokine/chemokine milieu during this experimentally induced disease [59,60]. Aggrecan core protein degradation is primarily mediated in cartilage tissue by the aggrecanases ADAMTS-4 and -5 [61,62]. ADAMTS proteins are thought to be produced primarily by chondrocytes and are activated following local and systemic production of inflammatory cytokines [63]. So, unlike spinal cord injury where CSPG up-regulation is detrimental, maintenance of aggrecan in joints is critical for preventing arthritis and therapies targeting aggrecanases are being proposed. 3.3. Multiple sclerosis In 2001, Sobel and Ahmed reported that CSPGs are up-regulated around the edge of active MS lesions but reduced in the centre of both active and chronic inactive plaques [17]; however, the biological significance of this altered expression beyond axonal regeneration is not yet clear. Specifically, whether the deposited CSPGs contribute to immunity is unknown. However, several studies have addressed the benefits of administering CSPGs as a therapeutic in the MS model, experimental autoimmune encephalomyelitis (EAE). When a CSPG GAG disaccharide breakdown product (CSPG-DS) is administered to EAE afflicted mice, they exhibit significantly reduced clinical disease scores [64,65]. This was determined to be due to a peripheral affect on T cells because CSPG-DS treatment resulted in a reduction of IFNγ and TNFα production by splenocytes [65]. Reduced T cell migration to IL-2 was also observed. However, when mice are treated with CS-A (a more complete CS GAG chain unattached to the proteoglycan core protein) they exhibit an exacerbated EAE disease course correlated with an increase in both T cell infiltration to the CNS and differentiation to the pathogenic Th1 and Th17 types [65]. Despite this, the precise mechanism of how CSPG-DS causes the inhibition of T cell differentiation and trafficking, and how CS-A promotes it, remains to be elucidated. It could be postulated that CSPG functioning through receptors such as CD44 is involved (see Section 4). Currently it is unknown whether CSPGs affect other peripheral immune cells such as macrophages, neutrophils and B cells, all of which have been implicated in EAE/MS. The above studies indicate that targeting CSPGs in EAE or MS may require a complete breakdown of CSPG products (as opposed to just cleaving the GAG chains from the core protein), or a reduction in their biosynthesis. Despite this, there remain many unanswered questions as to the role of CSPG up-regulation around MS lesions in the CNS,

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which CSPG member(s) is critical for regulating immunity, as well as effects of CSPG distribution changes in the CNS on infiltrating encephalitogenic and non-antigen-specific cells. Considering the wealth of information regarding the damaging effects of altered CSPG expression in spinal cord injury models it is promising to speculate that CSPGs play a significant role in lesion generation and lack of CNS repair during MS/EAE. Furthermore, it is tempting to postulate that reducing CSPGs in the CNS of MS patients may decrease neuroinflammation, and encourage neuroregeneration and clinical improvement. 3.4. The benefits of CSPG expression in pathology On the surface, CSPG expression appears to be detrimental in almost all pathologic conditions in the CNS due to its role in blocking neurite outgrowth and therefore repair. Past research has overwhelmingly demonstrated that CSPG-mediated blockade of axonal outgrowth is one of the most damaging components of the glial scar [12–16,31]. However, when cultured microglia are treated with CSPG-DS they take on a more regulatory phenotype, indicating that CS disaccharides may have an important role in immune regulation in the CNS [66]. It is also becoming apparent that the glial scar, which forms immediately following injury to the spinal cord and includes high levels of CSPGs, may have beneficial aspects, particularly at early stages post-injury [39,67,68]. In 2008 Rolls and colleagues reported that when xyloside (see Section 2.1) is administered immediately at the time of experimental spinal cord injury, functional recovery is worsened; whereas when xyloside is administered two days following injury the prognosis is improved [39]. This was suggested to be due to an important role for CSPGs in the activation of macrophage/microglia to create a barrier that contains damage to the injured area. It was acknowledged that CSPG ablation following the initial window post-injury, where CSPGs play a neuro-protective role, would still be beneficial. Therefore, the careful timing of therapeutic approaches to reducing CSPGs in the injured or diseased CNS will be essential. 4. A co-conspirator? CSPGs and their direct/indirect interaction with peripheral immune cells There are several mechanisms by which CSPGs can interact with the immune system. Firstly, GAG chains on CSPGs can bind to both homeostatic (SLC) and inflammatory chemokines (IP-10, SFD-1β, MCP1, RANTES, PF-4) [2,5]. The capacity of CSPGs to bind to these proteins is most likely due to a physicochemical static attraction to the GAG chains as opposed to a highly-specific, epitope-dependent, mechanism. Binding of chemokines to CSPGs can thus regulate the trafficking of immune cells into the CNS to promote immune reactivity. CSPGs have also been widely demonstrated to be able to bind to and activate the CD44 receptor [29,39], which itself has sites on which chondroitin sulphate GAG chains bind (and therefore help regulate the function of CD44 as well as enhance cell activation [69]). CNS-resident microglia cells are directly activated by CSPGs via the CD44 receptor and blockade of CD44 results in reduced activation and modulated neurotrophic factor secretion [39]. Furthermore, a CD44-like CSPG cell surface protein has been implicated as being significantly responsible for invasion of cancer cells, particularly in melanomas. This suggests that CSPG proteins can enhance cell migration [70] (which is also possible through their ability to bind L- and P-selectin [29]). Therefore, through this interaction with CD44, which is widely expressed on leukocytes and lymphocytes, CSPGs may have a previously underestimated impact on these cell types. Aside from the abovementioned interactions that CSPGs have with the immune system, they are also able to directly influence cell differentiation. When CSPG disaccharide subunits (CSPG-DS) are injected to mice suffering from EAE, the immune system is driven

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away from the Th1- and Th17-types, whereas when the longer CS-A GAG chains are injected T helper cell differentiation to these pathogenic types is exacerbated [64,65]. CSPG-DS is also capable of reducing T cell trafficking towards SDF-1α in vitro [64]. As mentioned in Section 3.2, animals elicit a strong cell-mediated anti-CSPG response which exacerbates and drives the autoimmune response during arthritis [59,60]. High levels of anti-aggrecan antibodies have also been observed in humans (reviewed in [59,71]); therefore, even though it is a self-antigen, it seems that the immune system is capable of generating a strong cell-mediated response against CSPG proteins during autoimmunity in the periphery, at least in animal studies [59,65]. Whether this is the case in CNS autoimmunity (such as MS) or not remains to be elucidated. Chondroitin sulphate chains are structurally similar to other proteoglycan-binding GAGs that constitute the ECM, such as heparan sulphate and dermatan sulphate, as well as hyaluronan (a large, non-sulphated GAG chain that is one of the chief components of the CNS ECM). Due to the structural similarity of chondroitin sulphate, dermatan sulphate, heparan sulphate and hyaluronan it is not unreasonable to postulate that these GAG chains may share functional similarities. Like chondroitin sulphate, both heparan sulphate and dermatan sulphate chains have been shown to be capable of enhancing CD44-mediated cellular activation and function [3,29,72–74]. Primarily through this interaction with CD44, and an interaction with several different growth factors (particularly fibroblast growth factor (FGF) [75,76]) and selectins (L-selectin and P-selectin [3,29,72,74]), heparan sulphate and dermatan sulphate are likely to have important roles in activation, adhesion and/or migration of immune cells. Like chondroitin sulphate, both dermatan sulphate and heparan sulphate GAG chains can also bind to inflammatory chemokines, as well as cytokines [3,4]. Heparan sulphate is also capable of binding to the receptor PTPσ, although HSPGs, which promote neuronal extension, and CSPGs appear to have opposing effects [77]. Hyaluronan has been shown to be very important for early inflammatory processes, through binding cytokines and chemokines, and activating T cells, neutrophils, mast cells, macrophages and eosinophils in a CD44- or toll-like receptor-dependant manner (reviewed in [74,78]). Endothelial cells stimulated with proinflammatory cytokines (such as TNF-α) up-regulate hyaluronan which facilitates early-adhesion of leukocytes to the endothelium [74,79]. Hyaluronan also accumulates in MS lesions and contributes to failure of remyelination in EAE by preventing oligodendrocyte precursor cell maturation [80]. Therefore, due to the structural similarity and the fact that these proteins/GAGs seem to share many functions, it is not implausible to assume that, in some instances, there may be some redundancy between proteoglycan-bound chondroitin sulphate, heparan sulphate, dermatan sulphate or hyaluronan. It may be elucidated in the future that CSPGs share many of these functions and may have more of a capacity to activate immune cells than has previously been estimated. 5. Looking to the future: manipulation of the CNS ECM for therapeutic benefit? As discussed in Section 2.1, several pharmacologic methods have been utilised to alter the expression of CSPGs in the CNS. There are advantages and limitations to each of these methods. Chondroitinase ABC is currently the most commonly used approach of reducing the CSPG burden in models of CNS pathology. Whilst chondroitinase ABC treatment may improve neuro-repair/functional recovery in vivo [37] and axonal outgrowth both in vitro and in vivo [21,37,54], limitations to its therapeutic use in humans include: chondroitinase ABC has a short-lived enzymatic activity; it leaves behind core proteins which therefore may have residual biological activity; and, because chondroitinase ABC is a product of P. vulgaris, immune reactions may develop to its use [49]. Xylosides have also been used widely to reduce the harmful effects of CSPG deposition in the injured CNS. Unlike chondroitinase ABC which breaks down CSPGs that have already been deposited, xylosides reduce

CSPG biosynthesis. Indeed, xylosides stop GAG chains from being attached to the linkage region during CSPG production, and they also reduce the secretion of the proteoglycan core protein to the ECM [35]. A limitation of xylosides, however, is that the molecules they target are also necessary for the biosynthesis of other proteoglycans, including heparan sulphate and dermatan sulphate proteoglycans; therefore, xylosides could reduce the production and deposition of other proteins necessary for ECM stability. In view of this non-selectivity of targets, inhibiting enyzmes specific for chondroitin sulphate biosynthesis, such as chondroitin synthase [81] and chondroitin pylomerizing factor [49], may prove to be a more specific approach to regulating CSPG biosynthesis. While the GAG chains of CSPGs have been demonstrated to be important when considering neuro-repair inhibition, the core protein of CSPGs also contain domains inhibitory for neurite outgrowth [82–84]. Thus, the inhibition of production of core protein may also be advantageous. Alternately, employing enzymes that degrade CSPG core proteins at lesion sites, such as by the use of ADAMTSs, may also prove beneficial to reduce the CSPG burden. 6. Conclusion It is clear that CSPGs play an important role in CNS physiology and pathology. Despite promising advances in CSPG-directed therapy, altering CSPG expression in pathologic conditions will need to be studied in depth in animal models before human trials are considered. Reducing the beneficial effects of CSPGs following injury or during disease, and breaking down or altering levels of existing CSPGs (e.g. in joints and perineuronal nets), could have detrimental long-term side-effects. However, due to the inhibitory and damaging properties of up-regulated CSPGs at lesion sites following damage to the CNS as well as their impact on cells of the immune system (as demonstrated in both MS and arthritis models), further advances in this field, particularly when considered as a sitedirected and/or combination therapy, will hopefully provide a significant opportunity to improve the outcome for patients with CNS-specific injuries or diseases (such as MS) where CSPGs act as a barrier to neuroregeneration and repair. 7. Take-home messages • • • •

CSPGs inhibit repair of the damaged CNS CSPGs have been shown to be up-regulated around MS lesions In EAE, CSPGs influence T cell activation and differentiation CSPGs can modulate immune cells both in the periphery and in the CNS • Therapeutic intervention to CSPGs may prove useful in CNS disease and injury Acknowledgements The studies of proteoglycans and proteases in the Yong laboratory are funded by operating grants from the Canadian Institutes of Health Research and the Multiple Sclerosis Society of Canada. SHJ is funded by a postdoctoral fellowship, and MK and LL by studentships, from the Multiple Sclerosis Society of Canada. References [1] Bignami A, Hosley M, Dahl D. Hyaluronic acid and hyaluronic acid-binding proteins in brain extracellular matrix. Anat Embryol (Berl) 1993;188:419–33. [2] Hirose J, Kawashima H, Yoshie O, Tashiro K, Miyasaka M. Versican interacts with chemokines and modulates cellular responses. J Biol Chem 2001;276:5228–34. [3] Kawashima H, Atarashi K, Hirose M, Hirose J, Yamada S, Sugahara K, Miyasaka M. Oversulfated chondroitin/dermatan sulfates containing GlcAbeta1/IdoAalpha13GalNAc(4,6-O-disulfate) interact with L- and P-selectin and chemokines. J Biol Chem 2002;277:12921–30. [4] Lortat-Jacob H, Grosdidier A, Imberty A. Structural diversity of heparan sulfate binding domains in chemokines. Proc Natl Acad Sci U S A 2002;99:1229–34.

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The economic burden of systemic lupus erythematosus In a recent review Zhu et al. (Arthritis Care & Research 2011; 5:751-60) evaluated the economic burden of systemic lupus erythematosus (SLE). Authors made a PubMed research in April 2010 and selected 11 papers. In these papers economic direct and indirect costs of the disease were evaluated. The authors quantified an important economic burden, in terms of health care resource consumption (US $ 3,735 US $ 14,410) and loss of productivity (employment rate 35.8% - 55%). However a punctual evaluation of the economic burden of SLE is very difficult, due to the significative methodological differences in the studies considered. Luca Iaccarino