**2.2. Class 3 semaphorins**

injury [22, 24]. Studies in recent years have been focusing on finding new strategies for sustained CS down-regulation, based on the success of ChABC, to provide a sufficient time window for regeneration to occur. The expression of ChABC using lentiviral vector show a down-regulation of CSs for up to 8 weeks, and the animal demonstrates an enhanced axonal sprouting and a superior functional recovery in the forelimb after a cervical contusion injury

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

One of the mechanisms of CSPGs mediating their functions is through binding to receptors or growth factors. Contactin-1, Nogo receptors (NgRs), and RPTPσ are the identified CSPG receptors [27–29]. The transmembrane receptor RPTPσ binds to CSPGs neurocan and aggrecan in the CNS [28]. RPTPσ induces growth cone collapse *in vitro* [30]. Mice with RPTPσ knockout show a robust regeneration, with cortical spinal tract fibers extend for a long distance after a spinal hemisection [31] Blocking the binding of CSPGs to RPTPσ using membrane-permeable peptide mimetics promotes serotonergic innervation, and the animal demonstrates enhanced functional recovery after spinal cord injury [30, 31]. This membrane-permeable peptide mimetics hold a strong promise to modulate CSPG-mediated inhibition in replacing ChABC. Apart from RPTPσ, NgRs also bind to CSs. Double mutant of Ngr1/Ngr3 showed an enhanced axon regeneration in optic nerve crush injury [29]. Contactin-1 binds specifically to highlysulfated CS, CS-E, on the contrary to other CSPG receptor, the binding of CS-E to contactin-1 promotes neurite extension [27]. Neuro2a cells with recombinant expression of contactin-1 demonstrate extensive neurite sprouting when cultured on CS-E [27]. CSPGs also interact with other growth factors, chemokines, and guidance molecules in the developing brain. By doing so, they control the availability of these factors to cells. While fibroblast growth factor (FGF)-2, FGF-10, FGF-16, and FGF-18 bind to highly sulfated CS chains, guidance proteins such as slit2, netrin1, ephrin A1 and A5, semaphorin (Sema) 3A, 5A, and 5B bind to CS chains in a sulfation-

CSPGs in the CNS also aggregate into a macromolecular structure on the surface of neurons called perineuronal nets (PNNs) [33, 34]. PNNs are dense matrix structures formed by four families of brain ECM molecules, including CSPGs, hyaluronan, hyaluronan, and proteogly‐ can link proteins (HAPLNs) and tenascins [33]. PNNs wrap the neuronal surface and are crucial in controlling synaptic and neuronal plasticity in the developing and injury CNS [4, 35]. PNNs form toward the end of the critical period for plasticity, and the formation is activity dependent [36, 37]. Dark rearing from birth delays the formation of PNNs in the visual cortex [36]. Similar observation is also reported with whisker trimming from birth in the barrel cortex of mice [38]. ChABC treatment removes this layer of CSPG-enriched PNN matrix and reacti‐ vates plasticity in the adult CNS, this includes spinal cord injury, visual cortex plasticity, cuneate nucleus plasticity, and more recently, on memory enhancement [35, 39, 40]. PNNs mediate their functions, in part, through binding to other molecules such as chemo-repulsive molecule sema3A and soluble transcription factor Otx2 [41, 42]. Both Sema3A and Otx-2 bind to the PNNs via the highly sulfated CS-E. Upon binding, Otx-2 is internalized into the cells and regulates the gene expression for the maturation of PNN-positive parvalbumin neurons in the cortex [42, 43]. Binding of Sema3A to PNN-glycans potentiates the inhibitory properties

of PNN-glycans to the outgrowth of DRG neurons *in vitro* [41].

[25, 26].

dependent manner [32].

Semaphorins (Sema) are a family of axon guidance molecules during CNS development [44]. The family is divided into eight classes and only five out of these eight Sema are expressed in vertebrates. Unlike other types of vertebrate Sema which are either transmembrane or membrane-anchored, class 3 Sema (Sema3) is the only secreted Sema in vertebrates [45]. To date, Sema3-A, −B, −C, −E, and -F have been identified in an injured CNS [46]. They are produced by the meningeal fibroblasts migrating into the lesion area and up-regulated the expression of different members of Sema3 [46, 47]. The binding of Sema3 to its receptors, neuropilins or plexins, induces a strong growth cone collapse in DRG neurons [48]. Apart from acting through the corresponding receptors, Sema3 also mediate their function though binding to the PNNs. We have previously shown that Sema3A binds to PNNs and that this binding is mediated by a specific CS structure in the PNNs, CS-E. Blocking the binding of Sema3A to PNN glycans reduces the inhibition imposed by PNN to DRG neurons [41].

## **2.3. Tenascin-C and -R**

Tenascin (Tn) family has four members and two of them are expressed in the CNS, including Tn-C and Tn-R [49, 50]. They are both up-regulated after CNS injury [51, 52]. Tn-C is expressed by astrocytes, radial glia, and subsets of developing retinal and hippocampal neurons during early CNS development. In adults, Tn-C is restricted to areas of high plasticity including the olfactory bulb, the cerebellum and the retina. Tn-C interacts with other ECM molecules such as integrins, proteoglycans, and collagen [50]. Tn-C up-regulates after CNS injury and interacts with the different CSPGs in the glial scar. This interaction has been implicated to the failure of axon growth beyond the injury site [53, 54]. Expression of an appropriate integrin isoform, which binds and uses Tn-C as substrate, elicits an enhancement in regeneration [55, 56].

Tn-R is trimer which is expressed in both the developing and adult brains, primarily by neurons, including the horizontal cells from the retina [57]. In adults, Tn-R is found in the PNNs [58, 59]. The trimeric TnR interacts with the core protein in the CSPGs, consolidating the PNN structure [60]. Tn-R has negative influence on axonal growth [49]. Knockout mice of Tn-R, which forms disorganized PNNs, demonstrates enhanced motor recovery after spinal cord injury suggesting that 1) Tn-R is important for PNN structure and that PNNs dissolution enhances plasticity for functional recovery [61].

#### **2.4. Laminins**

Laminins are large heterotrimeric glycoproteins that contain an alpha chain, a beta chain and a gamma chain joined together in a coiled–coiled structure. Sixteen isoforms have been identified *in vivo*, and are differentially expressed both temporally and spatially in various tissues [62]. Genetic disruptions of laminin chains lead disruptions in various tissues and also functional properties in the CNS [63, 64]. The major receptors for laminins are classified as integrins and non-integrins [65]. We shall discuss the role of integrins in later sections of this chapter. For non-integrins receptors such as dystroglycan and GM1 gangliosides, the binding of laminin serves critical functions in the peripheral nervous system (PNS) including myeli‐ nation by Schwann cells, neurite outgrowth, and the integrity of blood–brain barrier [66–70]. In the CNS, laminin is primarily present in the basement membrane and is up-regulated by astrocytes after injury [71], although reports of individual isoforms of laminin have also been reported [72]. This suggests an neuronal heterogeneity of laminin isoforms in the adult brain.

Laminin provides a positive guidance to axons during development [73], and act as a sup‐ porting substrate to adult retinal ganglion cell [74] and retinal pigment epithelial cells *in vitro* [75]. The expression of laminin decreases during maturation of the optic system [76] even though, in our recent study, we observed that laminin is still present in adult retinas and optic nerves [74].

## **2.5. Collagens**

Collagens are the most abundant proteins in the animal kingdom, there are now 29 known collagens [77, 78]. A triple-helical organization of component pro- α-chains defines the collagens and 49 distinct collagen α-chain gene products have been described [79]. Collagens are classified into both fibrillar and non-fibrillar forms and can also be assembled into reticular networks and sheets [80]. The organization, distribution, and density of fibrils and networks vary with tissue type and the direction and magnitude of forces to which are given tissue is subjected.

Collagens expressed in the PNS provide a scaffold for the growth and attachment of Schwann cells which also guide the neurite extension [78, 81]. After injury, there is an over up-regulation of collagen which changes the mechanical properties of the lesion area, hinders, and delays regeneration to occur [78]. Collagen is implicated in the progression of glaucoma, a visual neurological disease. One of the characteristics of certain glaucoma is the increment of the intraocular pressure in the anterior chamber of the eye. The heightened pressure is transmitted to the posterior eye chamber, pressing the retinal ganglion cells and eventually driving them to death [82]. Since these cells are the neurons responsible for transmitting the visual signal from the eye to the brain, their cell death leads to inevitable blindness. One of the reasons for the increasing pressure in the anterior chamber is due to an obstruction of the filtering tissue present in the trabecular meshwork, where the aqueous humor flows. The cells of this tissue have the ability to secrete the extracellular matrix. In an attempt to adapt to a biomechanical insult, the cells in the trabecular meshwork increase the synthesis of ECM, including collagen, thus blocking the flow of the aqueous humor and leading to an elevation of intraocular pressure. This mechanism has been proposed as possible cause of the origin of glaucoma [82, 83].

Other important implication of collagens in glaucoma is found in the lamina cribosa, a structure located in the optic nerve head where axons exit from the retina to the optic nerve [84]. A dysregulation in collagen secretion at this point implicates an interruption of axonal transport from the retinal ganglion cell in the retina to the visual areas in the brain. Studies have shown that activated astrocytes are cells responsible for the collagen synthesis and alterations here [85].

The third implication of collagen in glaucoma is the stiffness of the sclera and its implication in the lack of elasticity of the eye [86]. The sclera is the structure that supports the attachment of ocular components including the retina and optic nerve head. A complex network of collagen fibers forms the sclera´s major component and is a major influence on the tissue´s biomechanical response to changes in the intraocular pressure. It has been proposed that the mechanical influence of the sclera may be a key on the eye injury after elevation of the intraocular pressure due to the alterations in the thickness, mechanics and matrix ultrastruc‐ ture provided by the arrangement of scleral collagen and fiber orientation [87].

### **2.6. Integrins**

nation by Schwann cells, neurite outgrowth, and the integrity of blood–brain barrier [66–70]. In the CNS, laminin is primarily present in the basement membrane and is up-regulated by astrocytes after injury [71], although reports of individual isoforms of laminin have also been reported [72]. This suggests an neuronal heterogeneity of laminin isoforms in the adult brain.

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

Laminin provides a positive guidance to axons during development [73], and act as a sup‐ porting substrate to adult retinal ganglion cell [74] and retinal pigment epithelial cells *in vitro* [75]. The expression of laminin decreases during maturation of the optic system [76] even though, in our recent study, we observed that laminin is still present in adult retinas and optic

Collagens are the most abundant proteins in the animal kingdom, there are now 29 known collagens [77, 78]. A triple-helical organization of component pro- α-chains defines the collagens and 49 distinct collagen α-chain gene products have been described [79]. Collagens are classified into both fibrillar and non-fibrillar forms and can also be assembled into reticular networks and sheets [80]. The organization, distribution, and density of fibrils and networks vary with tissue type and the direction and magnitude of forces to which are given tissue is

Collagens expressed in the PNS provide a scaffold for the growth and attachment of Schwann cells which also guide the neurite extension [78, 81]. After injury, there is an over up-regulation of collagen which changes the mechanical properties of the lesion area, hinders, and delays regeneration to occur [78]. Collagen is implicated in the progression of glaucoma, a visual neurological disease. One of the characteristics of certain glaucoma is the increment of the intraocular pressure in the anterior chamber of the eye. The heightened pressure is transmitted to the posterior eye chamber, pressing the retinal ganglion cells and eventually driving them to death [82]. Since these cells are the neurons responsible for transmitting the visual signal from the eye to the brain, their cell death leads to inevitable blindness. One of the reasons for the increasing pressure in the anterior chamber is due to an obstruction of the filtering tissue present in the trabecular meshwork, where the aqueous humor flows. The cells of this tissue have the ability to secrete the extracellular matrix. In an attempt to adapt to a biomechanical insult, the cells in the trabecular meshwork increase the synthesis of ECM, including collagen, thus blocking the flow of the aqueous humor and leading to an elevation of intraocular pressure. This mechanism has been proposed as possible cause of the origin of glaucoma [82,

Other important implication of collagens in glaucoma is found in the lamina cribosa, a structure located in the optic nerve head where axons exit from the retina to the optic nerve [84]. A dysregulation in collagen secretion at this point implicates an interruption of axonal transport from the retinal ganglion cell in the retina to the visual areas in the brain. Studies have shown that activated astrocytes are cells responsible for the collagen synthesis and

nerves [74].

subjected.

83].

alterations here [85].

**2.5. Collagens**

Integrins are a family of cell surface receptors that are important for cell adhesion to ECM proteins. They are the principal receptors on animal cells for mediating most ECM attachment and signaling. They connect the extracellular environment to intracellular cytoskeleton and are responsible for the activation of many intracellular signaling pathway [88]. All integrins are heterodimeric molecules containing two subunits, α and β. Each αβ combination has its own specificity and signaling properties [89]. Most integrins recognize several ECM proteins. Conversely, individual matrix proteins, such as fibronectin, laminins, collagens, and vitronec‐ tin bind to several integrins [90, 91].

The binding of ECM to integrins is regulated by integrin conformation which is determined by the activity inside the cell (inside-out signaling), while upon binding to the ECM molecule, integrin also changes its conformation and elicits signals that are transmitted into the cell (outside-in signaling) [92]. The best understood binding site for integrins is the Arg-Gly-Asp (RGD), which is also found in fibronectin, vitronectin, tenascin, and other ECM proteins. There are 24 types of integrins in humans, formed by the 18 different α-chains and 8 different βchains, dimerized in different combinations [88]. Each integrin dimer has distinctive properties and functions. Moreover, because the same integrin molecule in different cell types can have different ligand-binding specificities, it is likely that additional cell-specific factors interact with integrins to modulate their binding activity [93]. One example of the variability in integrin expression is in the adult retinal ganglion cells. Cells growing on different ECM *in vitro* express several distinct combinations of integrins although they are activated and signaled through the focal adhesion kinase pathway [74].

We and the others have previously shown that integrin activation encourages glial cells or neurons to traverse inhibitory areas. Non-specific activation using manganese or specific beta-1 integrin activating antibody, we were able to promote the migration of Schwann cells over CSPG substrate *in vitro* and encourage axonal outgrowth of DRG neurons [94, 95]. Similarly, integrin activation by over-expressing an integrin mediator kindlin-1 encourages axonal extension on CSPG substrate in DRG neurons *in vitro* and the growth of DRG neurons passing the lesion site into the spinal cord *in vivo* [96].

In visual system, integrins are equally important in mediating the cellular response to ECM. Integrin activation using manganese or beta-1 integrin activating antibody enhances the attachment of retinal pigment epithelial cells (RPE) on pathological Bruch's membrane, which contains a high level of Tn-C, in aged macular degeneration [55]. In retina, we and the others have also shown that adult retinal neurons have the capacity to grow on multiple ECM substrates including collagen I, collagen IV, fibronectin and laminin with different affinity. This differential binding influences the degree of branching and elongation of neurites [75]. Moreover, we demonstrated that much of effects act through integrins activation.
