**3.2. Astrocytic fibronectin in CNS disease pathology**

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

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

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

altered ECM, is thought to enhance the demyelination seen in this disease [129].

role of this integral ECM protein in spinal cord injury.

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

Inflammation-mediated loss of myelin and incomplete remyelination are pathological hallmarks of multiple sclerosis (MS). Remyelination is essential for both restoration of saltatory conduction and axonal protection [132]. Although remyelination occurs in the early stages of MS, it declines as the disease progresses, resulting in chronically demyelinated plaques and axonal loss [133]. Oligodendrocyte progenitors, the cells responsible for CNS remyelination [134], are present in most MS lesions, but ultimately fail to differentiate into mature myelinat‐ ing oligodendrocytes, which results in remyelination failure [132, 135]. One of the many factors regulating the migration, proliferation, and differentiation of oligodendrocyte progenitor cells into mature, myelinating oligodendrocytes is the extracellular matrix [136]. In multiple sclerosis brain tissue, enhanced fibronectin deposition was primarily localized to vessel walls, in particular in perivascular infiltrates, and correlated with the extent of inflammation. Fibronectin accumulation was also detected in the parenchyma of active demyelinating MS lesions, suggesting that, in addition to extravasation from affected blood vessels, fibronectin may be locally produced by endothelial cells, infiltrated macrophages in the CNS [15, 137], as well as astrocytes [14]. Recent data have now demonstrated that fibronectin inhibits the differentiation of oligodendrocyte progenitors and, as a result, remyelination [138]. This finding was furthered when Stoffels et al. observed that the production of fibronectin aggre‐ gates inhibited oligodendrocyte progenitor cell differentiation in both an animal model of MS and within chronically demyelinated lesions. When they examined the expression of fibro‐ nectin on demyelinating injury, they found that the formation of these inhibitory fibronectin aggregates is mediated by inflammation. In toxin-induced lesions, fibronectin expression was transiently increased within demyelinated areas and declined as remyelination proceeded. However, upon the examination of chronically demyelinated MS lesions, fibronectin expres‐ sion persisted in the form of insoluble, degradation-resistant aggregates. This finding was also observed in a mouse model of MS, chronic experimental autoimmune encephalomyelitis, wherein fibronectin aggregates were found at the relapse phase but not in a toxin-induced demyelination injury model.

Frost et al. [139] showed that fibronectin promoted the migration of oligodendrocyte precursor cells. Connecting segment-1 fibronectin, an alternative splice variant of fibronectin, localized to astrocytes and astrocyte end-feet at the edge of MS lesions [16]. The CS-1 domain serves as a ligand for a4B1, a leukocyte integrin involved in cell-ECM and cell-cell adhesion. The presence of CS-1 fibronectin in astrocyte end-feet may therefore contribute to entry or retention of a4B1 integrin-bearing leukocytes further into the CNS parenchyma. These data indicate that fibronectin and its splice variants have an active part in MS lesion development and progres‐ sion.

Fibronectin has also been shown to mediate the inflammatory response in spinal cord injury. After spinal cord injury, both a glial and fibrotic scar forms at the site of injury. An excellent review on the glial scar can be found in Sofroniew and Vinters, 2010. Along with the reappearance of tenascin-c, fibronectin deposition is also increased following spinal cord injury. While fibronectin has been shown to be a growth-permissive substrate for axons, the fibrotic scar is inhibitory to axon regeneration [140]. In a compression trauma model of spinal cord injury, Farooque et al. found that fibronectin was present within sites of severe spinal cord compression trauma; however, when distal portions of the spinal cord were probed for fibronectin antigen, there were no signs of deposition [141]. This indicates that fibronectin is deposited proximal to the site of injury. Additionally, fibronectin has been shown to stimulate astrocyte proliferation through two means: (1) the α5β1 integrin found on astrocytes, and (2) the up-regulation of the P2Y1 receptor. The up-regulation of P2Y1 by fibronectin requires [Ca2+]i and the activation of the integrin-linked kinase (ILK) and Akt [142]. The [Ca2+]i stimulated by fibronectin is a5B1 integrin receptor dependent and the phosphorylation of Akt or extracellular signal-regulated protein kinase (ERK1/2) induced by fibronectin mediates the action of cAMP response element-binding protein (CREB) and signal transducer and activator of transcription 3 (Stat3). Through these various pathways, fibronectin release could stimulate the astrocyte proliferation seen after spinal cord injury, that the increased expression of the P2Y1 receptor would provide more sites for ATP to bind, which could further aggravate the proliferation and inflammation of spinal cord astrocytes, thus worsening the recovery of Spinal cord injury patients [142].

#### **3.3. Laminin in CNS disease pathology**

Laminins are high-molecular weight (~400 kDa) proteins of the extracellular matrix. They are a major component of the basal lamina (one of the players of the basement membrane), a protein network foundation for most cells and organs. The laminins are an important and biologically active part of the basal lamina, influencing cell differentiation, migration, and adhesion. Laminin is vital for the maintenance and survival of tissues. In the central nervous system, laminins, similar to other extracellular matrix proteins, are broadly expressed during embryonic brain development. In the adult brain, however, the distribution of laminin is restricted to the vascular basal lamina and the ventricular-subventricular zone stem cell niche. We will be covering laminin expression as it pertains to the vascular basal lamina (i.e., basement membrane). There are two distinct continuous basement membranes that can be identified surrounding the cerebrovasculature: the vascular basement membrane and the astroglial basement membrane. Both of the basement membranes are composed of the characteristic sheet-like structures of laminins, heparan sulfate proteoglycans, entactin, and type IV collagen. The only difference observed between the two basement membranes is the source of the structural components: endothelial cells are the predominate source for the vascular basement membrane, and astrocytes (specifically, astrocytic end-feet) are responsible for the formation of the astroglial basement membrane.

Frost et al. [139] showed that fibronectin promoted the migration of oligodendrocyte precursor cells. Connecting segment-1 fibronectin, an alternative splice variant of fibronectin, localized to astrocytes and astrocyte end-feet at the edge of MS lesions [16]. The CS-1 domain serves as a ligand for a4B1, a leukocyte integrin involved in cell-ECM and cell-cell adhesion. The presence of CS-1 fibronectin in astrocyte end-feet may therefore contribute to entry or retention of a4B1 integrin-bearing leukocytes further into the CNS parenchyma. These data indicate that fibronectin and its splice variants have an active part in MS lesion development and progres‐

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

Fibronectin has also been shown to mediate the inflammatory response in spinal cord injury. After spinal cord injury, both a glial and fibrotic scar forms at the site of injury. An excellent review on the glial scar can be found in Sofroniew and Vinters, 2010. Along with the reappearance of tenascin-c, fibronectin deposition is also increased following spinal cord injury. While fibronectin has been shown to be a growth-permissive substrate for axons, the fibrotic scar is inhibitory to axon regeneration [140]. In a compression trauma model of spinal cord injury, Farooque et al. found that fibronectin was present within sites of severe spinal cord compression trauma; however, when distal portions of the spinal cord were probed for fibronectin antigen, there were no signs of deposition [141]. This indicates that fibronectin is deposited proximal to the site of injury. Additionally, fibronectin has been shown to stimulate astrocyte proliferation through two means: (1) the α5β1 integrin found on astrocytes, and (2) the up-regulation of the P2Y1 receptor. The up-regulation of P2Y1 by fibronectin requires [Ca2+]i and the activation of the integrin-linked kinase (ILK) and Akt [142]. The [Ca2+]i stimulated by fibronectin is a5B1 integrin receptor dependent and the phosphorylation of Akt or extracellular signal-regulated protein kinase (ERK1/2) induced by fibronectin mediates the action of cAMP response element-binding protein (CREB) and signal transducer and activator of transcription 3 (Stat3). Through these various pathways, fibronectin release could stimulate the astrocyte proliferation seen after spinal cord injury, that the increased expression of the P2Y1 receptor would provide more sites for ATP to bind, which could further aggravate the proliferation and inflammation of spinal cord astrocytes, thus worsening the recovery of Spinal

Laminins are high-molecular weight (~400 kDa) proteins of the extracellular matrix. They are a major component of the basal lamina (one of the players of the basement membrane), a protein network foundation for most cells and organs. The laminins are an important and biologically active part of the basal lamina, influencing cell differentiation, migration, and adhesion. Laminin is vital for the maintenance and survival of tissues. In the central nervous system, laminins, similar to other extracellular matrix proteins, are broadly expressed during embryonic brain development. In the adult brain, however, the distribution of laminin is restricted to the vascular basal lamina and the ventricular-subventricular zone stem cell niche. We will be covering laminin expression as it pertains to the vascular basal lamina (i.e., basement membrane). There are two distinct continuous basement membranes that can be identified surrounding the cerebrovasculature: the vascular basement membrane and the

sion.

cord injury patients [142].

**3.3. Laminin in CNS disease pathology**

In multiple sclerosis lesions, alterations in the basement membrane are observed [16, 91, 143]. Abnormalities of the basal lamina surrounding the brain capillaries and local deposition of matrix molecules may influence blood-brain barrier permeability and thus leukocyte migra‐ tion and retention. The basement membrane barriers previously discussed—vascular and astroglial—define the inner and outer limits, respectively, of the Virchow-Robin perivascular space where leukocytes accumulate before they migrate into the CNS neuroparenchyma. Recently, the presence of extensive basement membrane alterations in MS brain tissue was described [16, 143]. It was found that inflammatory MS lesions contained irregular and discontinuous basement membranes throughout the lesion area. It was also found that organized deposition of basement membrane proteins occurred within the perivascular infiltrates in MS lesions. This group hypothesized that these structures contributed to the influx of leukocytes by forming a reservoir of chemotactic agents. However, they also posited that the perivascular ECM structures might function as a conduit network, thereby facilitating the transport of myelin-laden macrophages out of the CNS toward cervical lymph nodes [144]. The deposition of such compact parenchymal basement membrane deposits may have further consequences such as hampering axonal regeneration and outgrowth through the formation of an anatomical barrier, which could lead to the persistence of MS lesions.

It has also been demonstrated that the only laminin isoforms present in the vascular basement membranes are α4 and α5, whereas isoforms α1 and α2 were restricted to the astroglial basement membrane [145]. When investigating the expression of these laminin isoforms in the brain tissue of experimental autoimmune encephalomyelitis mice, leukocyte infiltration was associated with a pronounced loss of laminin α5 immunoreactivity in the vascular basement membrane. However, in regions where laminin α4 and α5 were detected, no leukocyte infiltration was detected. Interestingly, there was major leukocyte infiltration occurring at sites where the parenchymal basement membrane contained both the laminin α1 and α2 chains, isoforms produced primarily by the astrocytic basement membrane. This suggests that leukocyte migration across the astroglial basement membrane is markedly different compared to the migration observed across the vascular basement membrane [145]. There was also a recent study looking into the differential distribution of several laminin isoforms in control and MS brain tissue. In this study, the authors confirmed the previous finding that the vascular basement membrane is mainly composed of laminin-5,-8, and -10, whereas the astroglial basement membrane predominantly consists of laminin-1 and -2. However, in active demye‐ linating MS lesions, they observed leukocytes accumulating around vascular basement membranes containing laminin α5. In addition, disruption and loss of vascular laminin expression in active demyelinating lesions have been reported [146].

Laminin also plays a role in maintaining the integrity of the blood-brain barrier (BBB). The BBB is a dynamic network that regulates material exchange between the circulatory system and the brain parenchyma, which aids in homeostatic maintenance of the central nervous system [147]. In the context of central nervous system injury, BBB malfunction has been reported. The BBB is mainly composed of brain microvascular endothelial cells, astrocytic endfeet, pericytes, and the basement membrane, of which laminin is a key component. Astrocytes wrap around endothelial cells using their end-feet, and pericytes, which are sandwiched between endothelial cells and astrocytes, signal to both cell types. Recently, it has been shown that pericytes are necessary for the formation of the BBB during embryogenesis, and loss of pericytes leads to comprised BBB integrity and age-dependent vascular-mediated neurode‐ generation in adult mice, which suggests an important role for pericytes in BBB regulation. In a recent report, a group found that astrocyte laminin, by binding to the integrin α2 receptor, prevents pericyte differentiation from the BBB-stabilizing resting stage to the BBB-disrupting contractile stage, which helps to maintain the integrity of the BBB [148]. However, when astrocytic laminin was down-regulated using functional blocking antibodies and RNA interference, there were decreases in aquaporin-4 expression on astrocyte end-feet and decreases in tight junction protein expression. Further, in laminin knockdown animals, the lack of astrocytic laminin induced the differentiation of pericytes from the resting stage to the contractile stage. This loss of astrocytic laminins could be one of the major driving forces behind the leakiness of the BBB seen in many neurodegenerative diseases and CNS injuries.

#### **3.4. Vitronectin in CNS disease and injury**

Unlike the preceding extracellular matrix proteins, vitronectin has remained elusive in its functional role in central nervous system inflammation and injury. The earliest reports observed an enhancement of vitronectin expression in the blood vessel walls of active demye‐ linating lesions, in demyelinated axons, and on a small number of hypertrophic astrocytes. However, a negative role for vitronectin has not been found. In contrast, vitronectin has been shown to promote neurite outgrowth [149] and enhance astrocyte migration [150]. As vitro‐ nectin mRNA is almost undetectable in the normal adult brain, it might be synthesized by infiltrating leukocytes or derived from the plasma as a result of blood-brain barrier breakdown. In the EAE model of multiple sclerosis, vitronectin expression was shown to be enhanced, as well as contribute to the up-regulation of matrix metalloproteinases and activation of microglia [151]. Increasing research into the role of this under-studied extracellular matrix protein could provide clues as to its functional role in CNS inflammation.
