**3. ECM regulation of astrocytes**

chapter we will instead focus on the known role of tenascin-c in NPC differentiation into astrocytes. The tenascin gene family has recently gained increased attention with regard to glial development Owing to their late embryonic and early postnatal expression. Karus et al. have recently shown that TnC is capable of regulating the maturation of astrocytes during embryonic spinal cord development, primarily by orchestrating the responsiveness of NPCs to growth factors [27]. Within the developing brain and spinal cord, NPCs have been observed to initially generate neuronal cells. However, changes in the expression patterns of growth factor receptors result in the specification of astroglial cells. The expression of the epidermal growth factor receptor has been shown to be critical for normal astrocyte development [95]. During early embryonic stages, NPCs expressing Nestin, brain lipid binding protein (BLBP), and fibroblast growth factor receptors (FGFR) primarily generate neurons. Upon sustained fibroblast growth factor (FGF) signaling, these NPCs gain responsiveness towards epidermal growth factor (EGF). The expression of the EGF receptor is stimulated by TnC [27]. As a result, a rapid decline in neuron generation is observed in the embryonic spinal cord. Regardless of their location along the rostro-caudal axis of the developing spinal cord, the NPCs begin to express shared molecular markers with astrocytes, such as glutamate aspartate transporter (GLAST) and TnC [96]. Additionally, these cells begin to express additional markers such as S100β, aquaporin-4 [97], and fibroblast growth factor receptor 3 (FGFR3). Subsequently, the NPCs differentiate into GFAP-positive mature astrocytes, which are then often classified into fibrous white matter and gray matter astrocytes. Moreover, CSPGs and potentially TnC are

Astrocytes have been shown to play a prominent role in the developing central nervous system. Astrocytes contribute significantly in coordinating neuronal migration, axon guidance, and synapse formation [92]. This coordination is directed through deposition of specific extracel‐ lular matrix protein in the developing CNS—namely fibronectin and laminin. In an early report, Stewart and Pearlman observed fibronectin-like staining in the developing mouse cerebral cortex [99]. The temporal and spatial expression of fibronectin led them to posit that the transient appearance of fibronectin-like immunostaining in the zones that contain early cortical afferents suggests a role for Fn in forming the migratory pathway for the growth cones of these axons. In this role, it may be acting in concert with other extracellular matrix compo‐ nents such as hyaluronectin [100], glycosaminoglycans [101, 102], and laminin [103], which have been shown to have similar spatial distributions. The decline of fibronectin-like immu‐ nostaining that occurs as cortical development progresses may be a part of the change from the immature state, which supports profuse axon elongation in the CNS, to the mature state in which neurite outgrowth is quite limited. In addition to fibronectin deposition, astrocytes produce and secrete laminin, a key extracellular matrix guidance molecule in the developing brain. Laminin is synthesized and secreted by astrocytes both *in vivo* [103–107] and *in vitro* [108–112]. Astrocytic laminin is deposited into the ECM and fixed on the cell surface through binding to specific transmembrane receptors known as integrins [113–115]. The regionalization of laminin on the astrocyte surface is determined by the clustering of integrins, which are bound to the microfilaments, into macromolecular complexes known as focal contacts [116, 117]. It is this organization of laminin into specific patterns on the cell surface that provides directional cues to the elongating neurite [118–120]. Similar to fibronectin and TnC, the

involved in the maturation toward GFAP-positive astrocytes [98]**.**

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

Once established, the composition of the mature extracellular matrix is rather stable with little or no turnover of their components [24]. This stability is lost, however, when tissue damage results from injury, inflammation, or disease. Extracellular matrix degradation is triggered through inflammation, which results in changes to the matrix composition. Under these circumstances, the expression of various extracellular matrix molecules is highly up-regulated and major depositions are observed often marking lesion sites, in particular, in association with glial scar tissue formation. The freshly produced ECM components may be secreted by reactive astrocytes, oligodendrocyte precursors, microglia/macrophages, and eventually by meningeal cells. The lesion and consequent reactive processes induce a matrix accumulation that strongly resembles the "juvenile-type" of meshwork previously observed during early nervous system development. In many CNS diseases, it is becoming increasingly clear that some ECM molecules are aberrantly expressed and others cleaved into bioactive fragments known as damage-associated molecular patterns (DAMPs) or "alarmins" [121]. Through their ability to bind to different types of pattern recognition receptors (PRRs), these ECM molecules can influence the phenotype and magnitude of inflammation. Moreover, the enzymes and inflammatory mediators released by immune cells further degrade or alter the composition of the ECM. For the purposes of this book chapter, we will focus on the role of astrocytes in CNS injury and disease and how the extracellular matrix influences their response. We will highlight how the extracellular matrix proteins mentioned in the introduction could have profound effects on CNS injury and disease recovery by discussing their known roles.

#### **3.1. Tenascin-c influences on astrocytes in diseases of the CNS**

Tenascin-c can act as a DAMP, eliciting activation of innate immune cells via binding to a TLR-4 [122]. This was first demonstrated in a model of arthritis where inflammatory disease symp‐ toms in TnC KO mice resolved rapidly; conversely, TnC injection elicited joint inflammation. TLR-4 stimulation up-regulate TnC in macrophages so tenascin-c can act in an autocrine loop to amplify acute inflammation [122]. Although acute TnC expression is required for proper would healing [123], persistent expression can be detrimental; TnC is up-regulated in mice with Alzheimer's disease, and its deletion reduces neuropathology and inflammation [124]. TnC is an important factor in propagating chronic inflammation and could act in a similar manner after spinal cord injury.

After spinal cord injury, *de novo* synthesis of TnC occurs around the site within three days. Expression of TnC has been shown to persist for at least 30 days post injury in this model [125]. TnC is expressed by astrocytes in the lesion border, within the dorsal columns, and within the lesion epicenter. Interestingly, astrocytes cultured on TnC express fewer scar-related markers and proliferate less than astrocytes grown on control substrates [126], implying that TnC may restrict astrogliosis and scar formation after spinal cord injury. Additionally, *in vivo* work on spinal cord injury in tenascin-c knockout (KO) mice have shown that spontaneous recovery of locomotor functions after spinal injury is impaired in these animals when compared to wildtype mice. The impaired recovery was associated with attenuated excitability of the plantar Hoffmann reflex, reduced glutamatergic input, reduced sprouting of monaminergic axons in the lumbar spinal cord, and enhanced post-traumatic degeneration of corticospinal axons [127]. In a follow-up study using a model of lumbar spinal cord hemisection injury, global deletion of TnC was associated with enhanced axonal plasticity and growth into the lesion site. While these recent reports provide contrarian views to the role of TnC in the injured spinal cord, the precise mechanisms responsible for these changes have not been determined. In their review on extracellular matrix regulation in the healthy and injured spinal cord, Gaudet and Popovich suggest performing complementary gain-of-function experiments in wild-type mice and analyses of specific cellular and molecular pathways (e.g., inflammation) in tenascin-c KO mice [121]. Clearly the authors the state, consistent up-regulation of TnC after injury and its ability to bind/activate TLRs suggest that it is a candidate for controlling inflammation after spinal cord injury [121]. Further research will need to be performed in order to tease apart the role of this integral ECM protein in spinal cord injury.

TnC has also been implicated in globoid cell leukodystrophy (GLD), also known as Krabbe disease. GLD is a rare and often-fatal demyelinating disease caused by mutations in the galactocerebrosidase (*galc*) gene that results in the accumulation of galactosylsphingosine ("psychosine") [128]. Aberrant deposition of the extracellular matrix protein TnC has been observed in the brains of GLD patients when compared to age-matched control subjects. Elevated deposition and expression of TnC have also been observed in brain tissues from twitcher mice, an authentic mouse model of GLD [129]. The elevated TnC levels have been implicated in enhancing astrocyte responses to psychosine and astrocytic production of matrix metalloproteinase (MMP)-3, which activates microglial responses, inducing the formation of "globoid-like" cells in culture [129, 130]. This dysregulation of astrocytes, in part mediated by altered ECM, is thought to enhance the demyelination seen in this disease [129].

Expression of TnC is also aberrant in multiple sclerosis (MS). This chronic inflammatory and CNS demyelinating disease involves autoimmunity directed against myelin. A neuropatho‐ logical hallmark of MS is glial scarring, formed by reactive astrocytes. Multiple sclerosis lesions can be broadly defined as inactive, chronic active, and chronic. Inactive MS lesions and the center of chronic active lesions are characterized by few leukocytes and extensive glial fibrillary acidic protein immunoreactivity, indicative of astrogliosis. Within acute MS plaques, a significant loss of tenascin-c immunoreactivity has been observed, whereas tenascin-c was distributed throughout chronic MS plaques at levels similar to or greater than those seen in normal-appearing white matter. Particularly reactive astrocytes have been shown to strongly express TnC, and several reports have shown a correlation between TnC induction and acute inflammation, suggesting that enhanced tenascin-c expression might function as a defense mechanism to control the inflammatory reaction [22, 131]. However, the loss of TnC seen in acute MS lesions is consistent with inflammatory cell-mediated breakdown of the extracellular matrix, which may be a marker of blood-brain barrier breakdown and leukocyte extravasation. Matrix metalloproteinases, which can degrade tenascins, are probably a factor in this inflam‐ matory-mediated matrix breakdown. Such a breakdown in the TnC matrix might lead to a loss of matrix-cellular interactions, influencing the radial extension of the active lesion. Further‐ more, the expression and preservation of TnC in normal-appearing white matter beyond the plaque edge may negatively influence migration. The increase in TnC seen in association with a reactive astrocyte subpopulation in extensively demyelinated and subacute lesions and scar formation in chronic MS lesions might also inhibit repair. It suggests that reactive astrocytes continue to produce TnC, which leads to the eventual increase in levels seen in chronic plaques. This increased production and deposition of TnC would then actively inhibit and prevent the differentiation of oligodendrocyte progenitor cells into oligodendrocytes within the lesion, leading to the failure to remyelinate axons, which is seen in MS pathology.
