**1. Introduction**

Tissues are not made up solely of cells. A substantial part of Tissue volume is extracellular space, which is largely filled by an intricate network of macromolecules constituting the extracellu‐ lar matrix (ECM). The vertebrate extracellular matrix was once thought to serve mainly as a relatively inert scaffold to stabilize the physical structure of tissues. But It is now clear that the matrix has a far more active and complex role in regulating the behavior of the cells that contact

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it. Throughout the body, the ECM provides structure and organization to tissues through an intricately arranged scaffold comprised of a variety of secreted proteins and complex polysac‐ charides that are secreted locally and assembled into an organized meshwork in close associa‐ tion with the surface of the cells that produced them. Variations in both the relative amounts of the different types of matrix macromolecules and the way in which they are organized in the extracellular matrix give rise to an amazing diversity of forms, each adapted to the functional requirements of the particular tissue that influences their survival, development, migration, proliferation, shape, intercellular communication, and function. The extracellular matrix has a correspondingly complex molecular composition. Although our understanding of its organi‐ zation is still incomplete, there has been rapid progress in characterizing many of its major components.

In this chapter, we will focus on a select group of ECM proteins—tenascin-C, fibronectin, vitronectin, and laminin—and their patterns of expression and influence on the response and function of glia in the developing and adult central nervous system (CNS). We will then provide a detailed discussion on the differences in the patterns of expression of these factors to specific changes observed in the context of neurological diseases using studies that have pioneered this new approach to understanding the contributions of glia to injury and inflam‐ mation. About 20% of the total volume of the adult CNS is extracellular space [1, 2] that contains

**Figure 1.** Schematic summary depicting the diverse impacts of select ECM proteins on astrocytes. Image shown is of a murine glial fibrillary acidic protein (GFAP+) astrocyte (red; center).

highly organized ECM [3]. As in peripheral tissues, the ECM is composed of both interstitial and basement membrane proteins of the ECM family; however, in the CNS, the ECM compo‐ sition is remarkably different. Whereas the interstitial ECM of most peripheral tissues is enriched in collagen, laminin, and fibronectin, the ECM of the adult CNS is primarily a loose meshwork of hyaluronan, sulfated proteoglycans, and tenascin-R [4, 5]. The significance of these ECM proteins in the adult CNS has been extensively considered in recent reviews [6–8] and is beyond the focus of this chapter. Instead, we will focus on the Aforementioned ECM proteins and their significance for astrocyte function (**Figure 1**).

### **1.1. Fibronectin**

it. Throughout the body, the ECM provides structure and organization to tissues through an intricately arranged scaffold comprised of a variety of secreted proteins and complex polysac‐ charides that are secreted locally and assembled into an organized meshwork in close associa‐ tion with the surface of the cells that produced them. Variations in both the relative amounts of the different types of matrix macromolecules and the way in which they are organized in the extracellular matrix give rise to an amazing diversity of forms, each adapted to the functional requirements of the particular tissue that influences their survival, development, migration, proliferation, shape, intercellular communication, and function. The extracellular matrix has a correspondingly complex molecular composition. Although our understanding of its organi‐ zation is still incomplete, there has been rapid progress in characterizing many of its major

116 Composition and Function of the Extracellular Matrix in the Human Body

In this chapter, we will focus on a select group of ECM proteins—tenascin-C, fibronectin, vitronectin, and laminin—and their patterns of expression and influence on the response and function of glia in the developing and adult central nervous system (CNS). We will then provide a detailed discussion on the differences in the patterns of expression of these factors to specific changes observed in the context of neurological diseases using studies that have pioneered this new approach to understanding the contributions of glia to injury and inflam‐ mation. About 20% of the total volume of the adult CNS is extracellular space [1, 2] that contains

**Figure 1.** Schematic summary depicting the diverse impacts of select ECM proteins on astrocytes. Image shown is of a

murine glial fibrillary acidic protein (GFAP+) astrocyte (red; center).

components.

Fibronectin (Fn) is a high-molecular weight, insoluble glycoprotein dimer that consists of three types of repeating amino acid modules, named type I, type II, and type III [9]. The structure of Fn varies, depending on whether it is secreted into the plasma or synthesized by resident cells. The majority of plasma Fn is produced by hepatocytes and is detectable in human blood at a concentration of 300 μg/ml [10, 11]. In contrast, cellular Fn contains the alternatively spliced extra domain A and/or extra domain B (the nomenclature for humans; for rodents: EIIA and EIIIB). In addition, Fn has been shown to be a critical component in other ECM proteins, including heparin, collagen, and fibrin, and together these protein networks contribute to the formation of the ECM [9]. One of the main functions of Fn is to serve as a scaffold for cell adhesion and migration, which influences the regulation of cell proliferation and differentia‐ tion [9]. A myriad of small proteins, such as growth factors, have been found to support these functions of Fn when they accumulate in the Fn network. As a result, local concentrations of these small proteins are seen to increase. The Fn matrix has been found to be essential for normal embryonic development by studying Fn-knockout mice [12]. In healthy adult tissue, Fn is expressed at low levels. Transient Fn re-expression either through plasma leakage and/or synthesis from resident cells is a common "default" response to tissue injury, ranging from skin wounds to joint inflammation [13] and myelin degradation [14]. In the CNS, myelin damage (demyelination) elicits the production of a temporary Fn matrix [14–18]. In this injury scenario, the Fn matrix is a result of plasma leaking into the CNS parenchyma [15, 16], and cellular Fn is secreted by resident astrocytes, microglia, and endothelial cells [14]. The gener‐ ation of a temporary Fn scaffold comprised of both plasma and cellular Fn is a common response to tissue injury. We will discuss Fn re-expression during glial scar formation in multiple sclerosis (MS) and how clearance of the temporary Fn matrix is disturbed, which results in incomplete remyelination.

#### **1.2. Tenascin-C**

Tenascin-C (TnC) is a glycoprotein that is expressed in the ECM of various tissues where it has been found to regulate processes such as cell growth, migration, and adhesion during development, and represents 25% of the class of proteins that form the basic constituents of the brain ECM [19–22]. Tenascins are very large multimeric glycoproteins whose structure is well-conserved among vertebrates. TnC is built up in a modular fashion and consists of a cysteine-rich amino-terminus, EGF-like domains followed by fibronectin type III domains, and a carboxyterminal domain resembling fibrinogen-b [23]. Tenascin-C binds and interacts with a wealth of extracellular matrix and cell surface ligands [20], which is heavily mediated by Fn type III modules. Integrins, cell-surface heparin sulfate proteoglycans, and cell adhesion molecules of the immunoglobulin superfamily have been found to be the major cellular receptors of TnC [24]. TnC commonly binds to other ECM proteins, such as fibronectin, phosphacan, and, particularly, leticans. During CNS development, TnC is first expressed and accumulates around the fibrous processes of radial and Bergmann glial cells, which direct the migration of neuronal precursors during cortical and cerebellar development, respectively [25, 26]. During the later stages of development, TnC is expressed primarily by astrocytes, where it is thought to exert autocrine effects that regulate the proliferation of astrocyte progenitor cells [27]. TnC has also been found to modulate the stem cell compartment in the subventricular niche, where it is specifically enriched in the environment of mouse neural stem cell precursor cells (NSPCs) at embryonic day E14–E15 [28]. For example, TnC has been found to contribute heavily to the maturation of NSPCs [29], as well as the proliferation and maintenance of oligodendrocyte precursor cells [30–32]. *In vivo* and *in vitro* studies have demonstrated that TnC encodes for both permissive and inhibitory cues, which mediate neuron migration and axon growth and guidance by way of neuron-glia interactions [33–39]. Two to three weeks after birth, TnC expression decreases continuously, maintaining only a significant expression level in the neurogenetically active areas of the adult brain that encompass the subependymal zone and the hippocampus, as well as regions of plasticity in the hypothalamus [40–44].

#### **1.3. Laminin**

Laminins are major components of the basal lamina [45] and are also present in the ventricular zone (VZ) of the developing neocortex [46, 47]. Additionally, laminins were one of the first ECM proteins to be implicated in nervous system development as they were found to promote neurite outgrowth in an integrin-dependent manner [48–52]. During development, the extracellular matrix forms a basal lamina (BL) surrounding the brain and blood vessels throughout the CNS [53, 54]. In the neocortex, the BL at the pial surface is contacted by the end-feet of radial glial cells. A number of studies have shown how crucial the pial BL is for neocortical development. Removal of the BL leads to the detachment of radial glial cell fibers, which affects radial glial cell survival and proper cortical lamination [55–58]. Laminins have also been shown to promote the expansion, migration, and differentiation of neural stem cells (NSCs) *in vitro* [46, 59–67]. Mice lacking laminin α1 die during embryogenesis [68]; mice bearing a mutation that only affects the laminin α1 nidogen-binding domain survive until birth and display disruptions of the pial basal lamina as well as neuronal ectopias [69]. Additionally, laminin α1inactivation in a subset of cortical neurons has been observed to cause cortical lamination defects [70]. However, defects in the maintenance and/or differentiation of NSCs has not been reported in these mutants. *In vivo* evidence for a role of laminins in controlling NSC behavior comes from studies of their dystroglycan and integrin receptors. In human patients, mutations within enzymes that glycosylate dystroglycan have been shown to produce cortical neuronal ectopias [71, 72]. Mice lacking dystroglycan in the CNS or bearing mutations in dystroglycan glycosyltransferase display BL disruptions and neuronal migration defects [73–75]. Laminins have also been found to play a role in axonal guidance *in vivo* [76]. In mice, laminin α1 deficiency results in the abnormal branching of myelinated axons from the corpus callosum [70]. These mutants also show abnormal neuronal migration, impaired activation of integrin downstream effectors, such as focal adhesion kinase and paxillin, and disrupted AKT/GSK-3 signaling, which has been implicated in neurite outgrowth [77]. The exact mechanisms underlying these abnormalities remain unknown. In the CNS, oligoden‐ drocytes derive mainly from precursors residing in the ventral VZ and ganglionic eminences. They proliferate and migrate before becoming mature, myelinating cells [78]. Oligodendro‐ cytes do not have a basal lamina, although there exists some evidence that developing oligodendrocyte precursor cells can secrete low levels of laminin [79], which suggests oligo‐ dendrocytes may interact with outside sources of laminin. Oligodendrocytes myelinate axons through extending multiple cell processes capable of ensheathing numerous axons [80, 81]. Expression of laminins during development correlates with the onset of CNS myelination [80, 82], and varied degrees of defects have been found in white matter tracts of patients suffering from congenital muscle dystrophy [83, 84]. Mice lacking laminin α2 have a developmental delay in oligodendrocyte maturation, resulting in hypomyelination [85, 86]. The degree of developmental delay is region-specific, which may reflect different laminin α2 requirements [86].

#### **1.4. Vitronectin**

a carboxyterminal domain resembling fibrinogen-b [23]. Tenascin-C binds and interacts with a wealth of extracellular matrix and cell surface ligands [20], which is heavily mediated by Fn type III modules. Integrins, cell-surface heparin sulfate proteoglycans, and cell adhesion molecules of the immunoglobulin superfamily have been found to be the major cellular receptors of TnC [24]. TnC commonly binds to other ECM proteins, such as fibronectin, phosphacan, and, particularly, leticans. During CNS development, TnC is first expressed and accumulates around the fibrous processes of radial and Bergmann glial cells, which direct the migration of neuronal precursors during cortical and cerebellar development, respectively [25, 26]. During the later stages of development, TnC is expressed primarily by astrocytes, where it is thought to exert autocrine effects that regulate the proliferation of astrocyte progenitor cells [27]. TnC has also been found to modulate the stem cell compartment in the subventricular niche, where it is specifically enriched in the environment of mouse neural stem cell precursor cells (NSPCs) at embryonic day E14–E15 [28]. For example, TnC has been found to contribute heavily to the maturation of NSPCs [29], as well as the proliferation and maintenance of oligodendrocyte precursor cells [30–32]. *In vivo* and *in vitro* studies have demonstrated that TnC encodes for both permissive and inhibitory cues, which mediate neuron migration and axon growth and guidance by way of neuron-glia interactions [33–39]. Two to three weeks after birth, TnC expression decreases continuously, maintaining only a significant expression level in the neurogenetically active areas of the adult brain that encompass the subependymal zone and the hippocampus, as well as regions of plasticity in the hypothalamus [40–44].

118 Composition and Function of the Extracellular Matrix in the Human Body

Laminins are major components of the basal lamina [45] and are also present in the ventricular zone (VZ) of the developing neocortex [46, 47]. Additionally, laminins were one of the first ECM proteins to be implicated in nervous system development as they were found to promote neurite outgrowth in an integrin-dependent manner [48–52]. During development, the extracellular matrix forms a basal lamina (BL) surrounding the brain and blood vessels throughout the CNS [53, 54]. In the neocortex, the BL at the pial surface is contacted by the end-feet of radial glial cells. A number of studies have shown how crucial the pial BL is for neocortical development. Removal of the BL leads to the detachment of radial glial cell fibers, which affects radial glial cell survival and proper cortical lamination [55–58]. Laminins have also been shown to promote the expansion, migration, and differentiation of neural stem cells (NSCs) *in vitro* [46, 59–67]. Mice lacking laminin α1 die during embryogenesis [68]; mice bearing a mutation that only affects the laminin α1 nidogen-binding domain survive until birth and display disruptions of the pial basal lamina as well as neuronal ectopias [69]. Additionally, laminin α1inactivation in a subset of cortical neurons has been observed to cause cortical lamination defects [70]. However, defects in the maintenance and/or differentiation of NSCs has not been reported in these mutants. *In vivo* evidence for a role of laminins in controlling NSC behavior comes from studies of their dystroglycan and integrin receptors. In human patients, mutations within enzymes that glycosylate dystroglycan have been shown to produce cortical neuronal ectopias [71, 72]. Mice lacking dystroglycan in the CNS or bearing mutations in dystroglycan glycosyltransferase display BL disruptions and neuronal migration defects [73–75]. Laminins have also been found to play a role in axonal guidance *in vivo* [76].

**1.3. Laminin**

Vitronectin (Vn) is a multifunctional plasma and ECM glycoprotein with multiple domains for interactions with plasma proteins like thrombin, anti-thrombin III, and plasminogen activator inhibitor-1 [87]. Vn is primarily synthesized in the liver [88] and has affinity for different integrins expressed on T-cells, platelets, endothelial cells, and macrophages. Com‐ paratively little is known about the expression patterns of Vn during CNS development; however, a role for Vn in the induction of neurite outgrowth has been shown [89, 90]. In the normal adult CNS, vitronectin is localized mostly to blood vessels, with the exception of capillaries, suggesting that small amounts of vitronectin can be deposited in the CNS under normal conditions [91].
