Integrins as Receptor Targets for Neurological Disorders

Abstract

This review focuses on the neurobiology of integrins, pathophysiological roles of integrins in neuroplasticity and nervous system disorders, and therapeutic implications of integrins as potential drug targets and possible delivery pathways. Neuroplasticity is a central phenomenon in many neurological conditions such as seizures, trauma, and traumatic brain injury. During the course of many brain diseases, in addition to intracellular compartment changes, alterations in non-cell compartments such as extracellular matrix (ECM) are recognized as an essential process in forming and reorganizing neural connections. Integrins are heterodimeric transmembrane receptors that mediate cell–ECM and cell–cell adhesion events. Although the mechanisms of neuroplasticity remain unclear, it has been suggested that integrins undergo plasticity including clustering through interactions with ECM proteins, modulating ion channels, intracellular Ca²⁺ and protein kinases signaling, and reorganization of cytoskeletal filaments. As cell surface receptors, integrins are central to the pathophysiology of many brain diseases, such as epilepsy, and are potential targets for the development of new drugs for neurological disorders.

1. Introduction

Neuroplasticity is the ability of the nervous system to react with adaptive changes to intrinsic or extrinsic challenges such as epilepsy, trauma, and brain injury. In many cerebrovascular and neuronal diseases, in addition to intracellular compartment changes, alterations in non-cell compartments such as extracellular matrix (ECM) are recognized as an essential process. There has been recent interest in the possible role of adhesion molecules, particularly integrins as ECM receptors, in neurological disorders because they form an important link between the ECM and the intracellular cytoskeleton (CSK) and signaling molecules (Fig. 1). In the brain, ECM proteins are synthesized and secreted into the extracellular space in a mesh-like structure by neurons and glial cells. During development and mature nervous systems, and in wound healing, the interaction between ECM and its receptor integrin has a pivotal role in maintaining structural and functional neuroplasticity. ECM and integrin aberrations are likely to contribute to imbalanced synaptic function in epilepsy, Alzheimer’s disease, mental retardation, schizophrenia and other conditions in the brain. (Dityatev & Schachner, 2003; Dityatev et al., 2010; Gall & Lynch, 2004; Gall et al., 2003).

Figure 1. Schematic diagram for integrin-mediated signaling pathways.

Integrins mediate cell– extracellular matrix (ECM) and cell–cell adhesion events through binding ECM proteins such as fibronectin and transmembrane proteins such as cell adhesion molecules (CAMs) on adjacent cells. The ECM-integrin system consists of extracellular matrix (ECM) proteins, integrins and focal adhesion complex (FAC) that includes non-receptor tyrosine kinases (NRTK) and cytoskeleton (CSK) proteins. In this model, integrin clustering by multivalent ligands, including ECM proteins, induces recruitment of CSK proteins such as talin, vinculin, actin, tubulin, actinin, paxillin and tensin, and NRTK such as focal adhesion kinase (FAK) and Src to the focal contact. Integrin-linked kinase (ILK), phospholipase C (PLC), inositol trisphosphate (IP3), diglyceride (DAG), protein kiase A (PKA), PKC, PKG, Raf, GTPase (e.g. Ras, Cdc42, Rac, SOS and C3G), mitogen-activated protein kinase (MEK), extracellular signal-regulated protein kinases (ERK), c-Jun N-terminal kinases (JNK), as well as adaptor proteins such as Grb2, Crk, and Sos, are also recruited to the ECM-integrin binding site. Integrins also mediate crosstalk with other cell surface receptors such as growth factor receptor (GFR), cadherins and cell adhesion molecules (CAMs). ECM-integrin-FAC interactions play an important role in modulation of ion channels, synaptic transmission, and neuroplasticity that modulates neuronal cell proliferation, differentiation, apoptosis and migration. Integrins play an important role in pathological processes such as inflammation, wound healing, epileptogenesis, angiogenesis, and tumor metastasis.

Studies of integrin-initiated intracellular signaling have shown that integrins modulate Ca²⁺ and K⁺ ion channels, intracellular Ca²⁺ concentrations, cellular contractile properties, protein kinase activity, and growth factor receptors (Barouch et al., 2000; Chan et al., 2001; Danen & Yamada, 2001; Davis et al., 2001; Davis et al., 2002; Gui et al., 2006; Hynes, 1992; Martinez-Lemus et al., 2003; Porter & Hogg, 1998; Rueckschloss & Isenberg, 2004; Waitkus-Edwards et al., 2002; Wu et al., 2011a; Wu et al., 2001; Wu et al., 1998; Wu et al., 2010b; Wu et al., 2010c; Wu et al., 2008a; Wu et al., 2008b; Yang et al., 2010; Yip & Marsh, 1997). Furthermore, ECM-integrin-CSK interactions play crucial roles in gene expression, cell proliferation, migration and differentiation, and cell survivals (Hynes, 1992; Kim et al., 2011; Schwartz, 2001; Yamada & Miyamoto, 1995).

Since the discovery of the first integrin receptor for ECM protein fibronectin (FN) in 1986 (Tamkun et al., 1986), eighteen α-subunits and eight β-subunits have been identified (Hynes, 2002). Interestingly, there are at least 24 distinct integrins (Reichardt & Prokop, 2011) even though there are twenty-four α-subunit and nine β-subunit genes in the human genome (Venter et al., 2001). A total of over 50,000 papers are published on integrins so far. Fewer than 1000 papers are in the field of neuroscience as compared to ~6000 papers in cardiovascular system, indicating that integrins are not widely explored in the brain.

Integrins are necessary for neuronal cells to attach, spread, migrate, and extend processes on ECM molecules. Interactions of ECM-integrin-focal adhesion complex (FAC) including CSK proteins and intracellular signaling molecules in neuronal systems play an important role in synapse morphology and number, neuron-neuron and neuromuscular synaptic transmission, and neuroplasticity that modulates neuronal cell proliferation, migration, and differentiation (Venstrom & Reichardt, 1993). Historically, the role of the ECM and its receptors was viewed as largely structural molecules. However, there is emerging evidence that these proteins and receptors also perform signaling and regulatory functions in neuronal pathophysiological processes such as memory, inflammation, wound healing, epileptogenesis, angiogenesis, and tumor metastasis. Therefore, integrins as cell surface receptors are attractive pharmacological targets for designing new therapies for brain diseases (Clemetson & Clemetson, 1998; Cox et al., 2010; Davis, et al., 2001; Gall & Lynch, 2004; Horwitz, 1997; Jin & Varner, 2004; Lal et al., 2007; Millard et al., 2011; Reichardt & Prokop, 2011; Ross & Borg, 2001; Wu & Davis, 1998).

This review describes the neurobiology of integrins, pathophysiological roles of integrins in neuroplasticity, and the therapeutic implications of integrin targeted drugs for nervous system disorders.

2. Structure and Function of Integrins

2.1. Integrins in neurons

Integrins are α-β-heterodimeric transmembrane glycoprotein receptors that mediate cell–ECM and cell–cell adhesion events through binding ECM proteins such as fibronectin (FN) and transmembrane proteins such as neural cell adhesion molecule (NCAM) on adjacent cells. Integrins also have functional relationships with other membrane receptors such as ion channels and growth factor receptors. Integrins are expressed by many cell types, including neuronal cells, leukocytes, tumor cells, cardiac cells, skeletal muscle cells, and vascular cells (Table 1 and Figure 1). The α-subunit determines integrin ligand specificity, and the β-subunit is connected to CSK and intracellular molecules and thereby affecting multiple signaling pathways. The extracellular domains interact and bind to ECM in a divalent cation-dependent manner. Short cytoplasmic tails of integrin associate with FAC such as CSK proteins and protein kinases such as non-receptor tyrosine kinases (NRTK) (Davis, et al., 2001; Wang et al., 1993).

Table 1. Integrin subunits and their endogenous ligands in the nervous and cardiovascular systems.

Data compiled from: (Chan, et al., 2003; Chun, et al., 2001; Clegg, et al., 2003; Glukhova & Koteliansky, 1995; Moiseeva, 2001; Morini & Becchetti, 2010; Pinkstaff, et al., 1999; Ross & Borg, 2001; Velling et al., 1999; Wu, et al., 2010b)

	Neuron	Glia	Blood Vessels	Heart	Ligands
α1β1	+	+	+	+	CN, LN
α2β1	+	+	+		CN, LN, CHAD
α3β1	+	+	+	+	LN, FN, CN
α4β1	+	+	+	+	FN, CN, VCAM, OSP
α5β1	+	+	+	+	FN, OSP
α6β1	+	+	−/+	+	LN, invasin
α7β1	+	+	+	+	LN
α8β1	+	+	+		NPNT, FN, VN, TN
α9β1	+ &	+	+		TN, OSP, VCAM
α10β1				+	CN
α11β1				+	CN
αvβ1	+	+	+	+	VN, FN, OSP, agrin
αvβ3	+	+	+	+	VN, vWF, FN, CN, OSP, TSP, TN
α6β4		+	+		LN
αvβ5		+	+	+	VN, OSP, NPNT
αvβ6	+ (rat)				FN, NPNT, TN, TSP
αvβ8	+ (rat)	+			CN, FN, LN
α4β7	+ (mice)				MadCAM, VCAM,
αDβ2			+		VCAM, ICAM
αLβ2		+			ICAM, Fb
αMβ2		+			iC3b, Fb

CHAD: chondroadherin; CN: collagen; Fb: fibrinogen; LN: laminin; MadCAM: mucosal adhesin cell adhesion molecule; NPNT: nephronectin; OSP: osteopontin; TN: tenascin; TSP: thrombospondin; VCAM: vascular cell adhesion; VN: vintronectin; vWF: von Willibrand factor;

^&in post-synaptics (muscle).

Integrins are distributed ubiquitously with increased expression in the axon growth cone in the central nervous system (CNS). They are highly differentiated in the brain with region-specific and cell type-specific expression (Figure 2). The β1-integrin receptors were first identified as mediators of neurite outgrowth on ECM components (Reichardt & Tomaselli, 1991). Since then at least 14 of the 24 known integrin subunit combinations have been reported in nervous system: α1β1, α2β1, α3β1, α4β1, α5β1, α6β1, α7β1, α8β1, α9β1, αvβ1, αvβ3, αvβ6, αvβ8, α6β4 (Table 1 and Figure 2) (Chan et al., 2003; Chun et al., 2001; Morini & Becchetti, 2010; Pinkstaff et al., 1999; Renaudin et al., 1999; Yanagida et al., 1999). Different heterodimers of integrin have different affinities to the various ECM ligands and will be activated according to ligand and generate a transient or persistent signals. The predominance of α1β1, α3β1, α4β1, α5β1, α6β1, α7β1, and αvβ3 integrins in neurons suggests there are extensive interactions with collagens (CN), FN, laminin (LN), and vitronectin (VN). A single integrin heterodimer can often bind more than one ligand and vice versa. For instance, α3β1 can bind to CN, FN, and LN whereas at least 4 heterodimers containing the β1 subunit alone, α3β1, α4β1, α5β1 and αvβ1, have been identified as FN receptors (Table 1).

Figure 2. Integrin subunits distribution in different regions of central nervous system.

Data compiled from: (Chan, et al., 2003; Chun, et al., 2001; Glukhova & Koteliansky, 1995; Moiseeva, 2001; Morini & Becchetti, 2010; Pinkstaff, et al., 1999; Ross & Borg, 2001; Velling, et al., 1999; Wu, et al., 2010b). Individual integrin reported in one species perhaps not in others indicated in parentheses.

Some integrins will increase expression in specific physiological and pathological situations. α1-, α3-, and α4-integrin subunits are widely distributed in the developing brain, while α5- and α7-integrin subunits are distributed in the adult brain (Pinkstaff, et al., 1999). Immunohistochemistry studies show that α1-, α2-, α4-, α5-, β1-,β3-, and β4-integrins are increased in the hippocampus in rats with pilocarpine-treated epilepsy (Fasen et al., 2003).

2.2. Integrins in glial cells

In general, integrins control cell migration, proliferation, and differentiation in non-neuronal glial cells. Glial cells play a key role, via integrins, in the development and repair of the nervous system by providing scaffolds and nutrition for (migrating) neurons from astrocytes and radial glia, myelination for ensheathing neurons from Schwann cells in peripheral nervous system (PNS) and oligodendrocytes in CNS, and neuroimmunological protection from microglia. At least 16 of the 24 known integrin subunit combinations have been reported in glial cells (Table 1).

Different integrins show diverse function in glial cells. Integrin β1-subunit and reelin are required for normal morphological formation of radial glia cells and Schwann cell differentiation (Forster et al., 2002; Pietri et al., 2004). The α5-integrin is essential for glial proliferation and the α4-integrin contributes for survival. The loss of α4-integrin or α5-integrin did not affect glial cells migration and differentiation (Haack & Hynes, 2001). In addition, αvβ1 integrins mediates oligodendrocyte migration, α6β1 integrin facilitates oligodendrocyte proliferation and Schwann cells adhesion and migration, α1β1 integrin involves in remodeling in Schwann cells, αvβ3 integrin allows astrocytes to adhere to osteopontin following PNS or CNS injury and oligodendrocyte proliferation, αvβ5 correlates with astrocyte adhesion and oligodendrocytes differentiation and blockage of migration, and αvβ8 promotes astrocyte migration (Blaschuk et al., 2000; Colognato et al., 2004; Colognato & Tzvetanova, 2011; Dubovy et al., 2001; Fernandez-Valle et al., 1994; Milner et al., 1996; Milner et al., 1999; Milner et al., 1997; Stewart et al., 1997). Integrin function from neuronal and glial cells as an integrated system will be discussed more in the following sections of this review.

2.3. Extracellular matrix proteins

ECM proteins are synthesized and secreted in the brain by neurons and glial cells. Neuronal ECM proteins include CN, FN, LN, proteoglycans, hyaluronan, reelin, and tenascins. The ECM receptors include integrins, dystroglycan, syndecans, and glypicans. This review is focus only on integrins, the major cell surface receptors for ECM. Other ECM receptor targets are beyond the scope of this review. In double-immunolabeling, reelin and the α3-integrin subunit are colocalized in a dendrite and a dendritic postsynaptic spine of adult nonhuman primate cortex (Rodriguez et al., 2000). Colozalization between α6 and LN, and α5 and FN have also been reported in embryonic neural crest cells (Strachan & Condic, 2004). ECM resembles a mesh-like structure surrounding cell bodies, proximal dendrites and axon initial segments and plays physical roles by serving as a structural support element or adhesive substrate. In addition, ECM molecules are present in basal lamina in the capillary, postcapillary venules, and constitute an essential part of the blood-brain barrier (BBB) (Ballabh et al., 2004). ECM also provides a signaling environment by serving as substrates for cellular receptors, thus triggering or influencing signaling events across cell membranes and providing a niche and anchor for signaling factors, and therefore regulates the bioavailability of signals. Neuronal cells do not only respond to the matrix through integrins, but they actively contribute to ECM formation and reorganization.

Cells determine the composition and quality of ECM and matrix receptors through differential gene expression, glycosylation, and endocytic trafficking. Neuronal cells also release extracellular proteases enzymes, such as collagenolytic matrix metalloproteinases, a disintegrin and metalloproteinase with thrombospondin motifs and tissue plasminogen activator, to help restructuring the matrix and significantly influencing the bioavailability of signals in responding to conditional changes, or regulating the passage of growing or migrating cells (Reichardt & Prokop, 2011). LN, VN, reelin and chondroitin sulfates are reported to be involved in neuronal development (Dityatev et al., 2007; Heinrich et al., 2006).

Different integrin binding sites even in the same ECM proteins might have different functions. Many neuronal matrix proteins, including FN and CN, contain the arginine-glycine-aspartate (RGD) recognition site which can be recognized by the majority of β1-integrins. Application of RGD peptide interferes with cell-ECM adhesion and disrupts a wide variety of integrin-mediated intracellular activities. Other than RGD, the IIICS region of FN contains a 25–amino acid sequence, including the leucine-aspartate-valine (LDV) motif that binds the α4β1 integrin (Geiger et al., 2001). RGD and soluble FN can decrease voltage gated L-type Ca²⁺ currents (Ca_L) and dilate the arterioles, while LDV in FN and insoluble FN can increase Ca²⁺ currents and constrict the arterioles (D’Angelo et al., 1997; Waitkus-Edwards, et al., 2002; Wu, et al., 1998). These ECM effects may contribute to vascular regulation in stroke and brain trauma. Overall, ECM proteins have pivotal functions at the BBB and contribute to the processes of neuronal cell migration, axonal growth and myelination, and to the assembly and functional maintenance of the neuromuscular junction. ECM-integrin-FAC axis and its mechanisms have a crucial impact on nervous system development, maintenance, degeneration, and regeneration at all levels, acting through the regulation of cell adhesion and signaling, endosomal trafficking, CSK dynamics, and gene expression (Reichardt & Prokop, 2011).

2.4. ECM-Integrin-FAC axis

Different integrins may have distinct functions according to their ligand binding and type of intracellular signaling that is activated. Integrins can function as signaling receptors that transduce biochemical signals both into and out of cells (Clark & Brugge, 1995). Integrin activation by ECM ligands can be described as three main activation states of rest, intermediate affinity state and activated high-affinity state (Cox et al., 2010), or 3 steps as inactive, active and clustering (Clark & Brugge, 1995). Integrins are not constitutively active. In general, integrins on oncogenic variants could be constitutively activated. Some integrins can bind their ligands in a resting state and then be activated, whereas others require activation before binding. Other than ECM ligands, extracellular factors such as mechanical stress (e.g. blood pressure), divalent cation concentration (e.g. Mn²⁺), and interleukin signaling also lead to integrin activation. After activation, integrin clustering by multivalent ECM proteins induces recruitments of CSK proteins (ECM-integrin-CSK axis) including actin, vinculin, talin, paxillin and tensin as well as NRTK kinases such as focal adhesion kinase (FAK) and the Src kinase family to the focal contact point that forming into large multi-protein aggregates, termed cell-matrix focal adhesion complex (FAC). Mitogen-activated protein kinases (MEK), PLC-γ, and small GTPases rho, rac, and cdc42 as well as adaptor proteins such as Grb2 and Sos are also recruited to the ECM-integrin binding site after integrin activation (Figure 1). Ligand binding induces conformational changes in integrins and intracellular signaling, referred to as “outside-in” signaling. This intracellular signaling cascades activated by integrin activation can result in the cytoskeletal changes needed to modulate growth cone structure, neurite outgrowth and axon migration. This signal transduction has been also shown to result in protein phosphorylation, increased intracellular calcium concentration, activation of glutamate receptor, gene transcription, and the release of neurotransmitters (Clark & Brugge, 1995).

Intracellular signaling molecules (e.g. CSK and protein kinases) converge on the cytoplasmic domain of integrin tails after initial ligand occupancy. In turn, ion channel activation, CSK remodeling, and intracellular signaling activities in neuronal cells can control integrin expression, activate the high affinity state of integrin (clustering), and manipulate remodeling of the ECM and ECM binding affinity onto integrin in the plasma membrane. Therefore, populations of integrins are not static, but change dynamically across periods of activation in response to physiological and pathological events. These alterations are referred to as “inside-out” signaling through ECM-integrin-FAC axis (Clark & Brugge, 1995; Gall, et al., 2003; Morini & Becchetti, 2010; Wu, et al., 2001). The inside-out signaling also occurs in regulating synaptic structure, signaling, and various forms of neuronal plasticity. In the brain, integrins are concentrated at sites of synaptic contact and are critical for the formation, maturation, and maintenance of synaptic structure (Nishimura et al., 1998). Basal synaptic communication requires that the presynaptic and postsynaptic faces of the synapse communicate via adhesive and intracellular signaling events (Hoffman et al., 1998a; Schachner, 1997). Cleavage of integrin-ECM focal adhesive connections could be an early step in the formation of new synaptic configurations from inside-out signaling, followed by rapid formation of new focal contacts (Hoffman, et al., 1998a).

2.5. Integrin crosstalk with cell surface receptors

As one of the cell surface receptors, integrins mediate crosstalk with other cell surface receptors such as growth factor receptors and cell adhesion molecules (Figure 1). It is highly established that nerve growth factor (NGF) stimulates integrin-dependent axon outgrowth, but the mechanism connecting two systems is not well understood. Integrins and growth factor receptors may activate parallel intracellular signaling pathways including extracellular signal regulated protein kinases (ERK)-type MEK. Co-stimulation of integrins and epidermal growth factor (EGF) receptors activates Pyk2 and FAK to promote outgrowth of neurite via paxillin induced cytoskeletal changes (Ivankovic-Dikic et al., 2000). Integrin clustering transactivates several receptor tyrosine kinases (RTKs) including platelet derived growth factor receptor and EGF receptor. Thus, integrins may increase signals generated by growth factor receptors through its FAC.

Transforming growth factor beta-1 (TGF- β1) latency-associated peptide is a ligand for the integrin αvβ6, and αvβ6 integrin expressing cells induce spatially restricted activation of TGF-β1. Mice lacking αvβ6 integrin develop exaggerated inflammation (Munger et al., 1999). Interactions between α6-integrin and growth factor enable newly formed oligodendrocytes to survive in response to limiting concentrations of soluble growth factors (Colognato et al., 2002). The αvβ8 has been shown to promote the activation of TGFβ through binding to an RGD sequence in the TGFβ latency-associated peptide (Mu et al., 2002). Baron et al have demonstrated a physical association between platelet-derived growth factor (PDGF) and αvβ3 receptors on oligodendrocytes. PDGF stimulated a protein kinase C-dependent activation of αvβ3 integrin, which in turn induced oligodendrocyte proliferation via a phosphatidylinositol 3-kinase-dependent signaling pathway (Baron et al., 2002).

It has been reported that synaptic remodeling would require alterations to interaction between integrin and cell adhesion molecules. Integrin mediate cell-cell interaction through cell adhesion molecules such as neural cell adhesion molecule (NCAM), intercellular adhesion molecule (ICAM), and vascular-cell adhesion molecule (VCAM). The NCAM L1 functionally interacts with β1-integrins to potentiate neuronal migration toward ECM proteins such as FN through endocytosis and MEK signaling, and that impairment of this function by L1 cytoplasmic domain mutations may contribute to neurological deficits in the X-linked mental retardation syndrome CRASH (corpus callosum agenesis, retardation, aphasia, spasticity, and hydrocephalus) (Thelen et al., 2002). During neuronal injury and cytokine stimulation, microglia bind to their targets using αLβ2 and α4β1 through interaction with ICAM-1 and VCAM-1 and migrating to the site through αMβ2 (Clegg et al., 2003).

Cross-talk has also been documented between integrin and cadherins. N-Cadherins and integrins function in a coordinated manner to effectively mediate the cellular interactions essential for adhesion and development. A peptide resembling the juxtamembrane (JMP) region of the cytoplasmic domain of N-cadherin results in release of the NRTK Fer from the cadherin complex and its accumulation in the integrin-induced FAC, and results in inhibition of N-cadherin and β1-integrin function. A peptide that mimics the first coiled-coil domain of Fer prevents Fer accumulation in the integrin cytoplasmic domain and reverses the inhibitory effect of JMP (Arregui et al., 2000). These results show that integrin activation is necessary to influence the functional of other cell surface receptors and vice versa.

2.6. Integrin crosstalk with neurotransmitter receptors

Integrin could also have crosstalk with neurotransmitter receptors. It has been reported that α7β1 integrin colocalizes and physically interacts with LN-induced acetylcholine receptors (AchRs) clusters. LN through α7β1 facilitates the clustering of AChRs by a proteoglycan agrin in the post-synaptic membrane of skeletal muscle. Applying blocking antibodies to αv-, α7-, or β1-integrins inhibit the AchRs clustering activity of LN and agrin (Burkin et al., 1998; Burkin et al., 2000; Martin & Sanes, 1997). RGD-containing peptides or antibodies to the β3-integrin prevent a developmental increase in glutamate release and a concurrent shift in expression of postsynaptic N-methyl-D-aspartic acid (NMDA)-receptor subunits (Chavis & Westbrook, 2001). In rat hippocampal slices and synaptoneurosomes, a RGD peptide GRGDSP, leads to potentiation of NMDA-gated currents, and increases the slope and amplitude of the fast excitatory postsynaptic potentials (EPSPs) because of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) activation (Bernard-Trifilo et al., 2005; Lin et al., 2003).

Integrin ligands such as RGD peptides and FN via β1-integrin increase EPSP in the hippocampus could pass through two pathways. One of these pathways is through a Src kinase dependent NMDA and/or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors pathway. Src tyrosine kinase increases protein phosphorylation of NMDA receptor subunits NR2A and NR2B in synaptoneurosomes and acute hippocampal slices. Another pathway is through a Src tyrosine kinase independent pathway. This pathway is related to activation of NMDA receptor, phosphorylation of Thr²⁸⁶ site on calmodulin-dependent kinase II, and phosphorylation of Ser⁸³¹ site on AMPA receptors. The β1 integrin subunit or a mixture of function-blocking antibodies to α3, α5, and αv-integrin subunits block integrin ligand effects on synaptic responses. The γ-aminobutyric acid-A (GABA_A) receptor antagonist picrotoxin does not reduce the enhanced effects of EPSP by integrin ligands (Bernard-Trifilo, et al., 2005; Kramar et al., 2003; Lin, et al., 2003). In β1-integrin KO mice, expressions of AMPA and NMDA receptor subunits are normal. However, EPSPs at CA3–CA1 synapses are dramatically reduced, probably because of postsynaptic defect in AMPA receptor (Chan et al., 2006). These results show that hippocampal integrin activation is necessary to control the functional level of postsynaptic NMDA and AMPARs.

2.7. Integrin and Ca²⁺ signaling

Integrins modulate calcium signaling. The regulation of voltage L-type Ca²⁺ (Ca_L) channels by integrin-ECM interactions may play a role in the regulation of neuronal migration, neurite extension and synaptic remodeling. Growth cone extension requires a permissive range of intracellular Ca²⁺ concentration, whereas the frequency of Ca²⁺ waves and spikes controls the rate of axonal elongation/spreading (Becchetti & Arcangeli, 2010). Neuronal Ca_L channels in fresh isolated neuronal cells and heterologously expressed neuronal Ca_L channel isoforms in HEK-293 cells are potentiated by α5β1 integrin. This potentiation is regulated by an intracellular signaling pathway involving phosphorylation of α_1C C-terminal residues Ser¹⁹⁰¹ and Tyr²¹²² by using truncation and site-directed mutagenesis strategies (Gui, et al., 2006; Wu et al., 2010a). These sites are known to be phosphorylated by PKA and c-Src, respectively. Soluble RGD containing peptide ligands increase the expression of mRNAs for the neurotrophins brain derived neurotrophic factor, nerve growth factor, neurotrophin-3, the neurotrophin receptors TrkB and TrkC, and cfos at least in part through effects on calcium influx in adult hippocampus (Gall, et al., 2003). In this context, integrin-Ca_L channel interactions appear to be critical for the up-regulation of brain-derived neurotrophic factor mRNA in hippocampal neurons. Work in cerebellar neurons, immune cells, and epithelial keratinocytes suggests that a stable rear-to-front Ca²⁺ gradient is formed in cells during chemotaxis/migration, which is thought to be one of the causes of the different behavior of the front and rear side, with retraction and detachment of the trailing edge during cell movement cycle.

Integrin-Ca_L channel interactions could also play a major role in the responses of neurons and blood vessels to injury and repair (e.g. stroke). A series observations from our previous studies are that at least 3 different integrins regulate Ca_L, Ca²⁺ activated K⁺ channels and myogenic responses in vascular systems, and in heterologously expressed smooth muscle Ca_L in HEK cells through inside-out and outside-in signaling between integrin-FAC (Davis, et al., 2002; Gui, et al., 2006; Waitkus-Edwards, et al., 2002; Wu, et al., 2001; Wu, et al., 1998; Wu, et al., 2008b; Yang, et al., 2010). Both neuronal activity and vascular reactivity are modulated by integrin signaling through the generation or exposure of new integrin ligands from limited degradation of ECM and/or turnover of new integrins during wound healing (Davis et al., 2000; Gui, et al., 2006). For instance, successful axonal regeneration is highly correlated with the induction of integrins on the surface of peripheral neurons; therefore, peptides derived from integrin ligand have the potential to act as therapeutic agents for neuronal regeneration (Cox, et al., 2010; Meiners & Mercado, 2003).

2.8. Integrin in nervous system development

Integrins play a major role in the development of the CNS by stabilizing cellular contacts during cell migration, cell proliferation, and neurite extension. Integrin and FAC signaling molecules recruitment (e.g. GTPases Cdc42, Rac1 and CSK actin at leading edge), and integrin-ECM detachment/deadhesion (at trailing edge) and efficient attachment/adhesion have been reported to dynamically change their position at leading and trailing edges in migrating cells during development (Becchetti & Arcangeli, 2010; Huttenlocher & Horwitz, 2011). Lacking ECM components such as LN or FN, or lacking the β1-family of integrins, mice die during the early steps of embryonic development. The loss of α4, α5, or/and β1-integrins from the mice embryo results in abnormal lamination of the cortex and cerebellum due to disruptions of the basal lamina that separates the brain from the overlying mesenchyme, and results in severe perturbations of the peripheral nervous system, including failure of normal nerve arborization, delay in Schwann cell migration, survival, proliferation, and differentiation, and defective neuromuscular junction differentiation (Feltri et al., 2002; Graus-Porta et al., 2001; Haack & Hynes, 2001; Pietri, et al., 2004). These alterations are likely to reflect the roles of integrin receptors in regulating activation of MEK, Rac, and other signaling pathways (Campos et al., 2004).

In an in vitro inhibition assays with function-integrin antibodies in avian neural crest cells, cell spreading is essentially mediated by FN receptor αvβ1 and α8β1, migration involved α4β1, αvβ3 and α8β1 (Testaz et al., 1999). In LN receptor α6-integirn knockout (KO) mice, the cortical basement membrane is disrupted with alterations of LN deposition, ectopic neuroblastic outgrowths are found on the brain surface and in the vitreous body in the eye, and myelin-forming oligodendrocytes show increased cell death (Georges-Labouesse et al., 1998). α3-integrin KO mice show a disorganized cortex and defective neuronal migration (Anton et al., 1999). The α5β1 integrin as FN receptor has been implicated in regulating morphology of cortical development and dendritic spines, formation of synapses in neurons, as well as in mediating neurite outgrowth after injury. Embryos lacking the α5-integrin subunit die at embryonic day 10.5 before cerebral cortex development (Marchetti et al., 2010). No obvious phenotypes in the peripheral nervous system have been reported in mice lacking the αv or β8 integrins. However, expression of αvβ8 is required for normal vascular development in the CNS. Absence of either αv or β8 integrins in the CNS leads to cerebral hemorrhage, seizures, axonal degeneration and premature death (McCarty et al., 2005, Proctor, 2005 #1806). In the future, conditional mutation on more subtle genetic models for integrins, ECM proteins, and FAC molecules will allow a more detailed understanding on the highly complicated function of integrin-mediated adhesion in the development of nervous system.

3. Physiological and Pathological Roles of Integrins in Brain Behavior and Brain Disorders

Very little is known regarding the role of integrins in neurophysiological and pathological conditions. Emerging evidence shows that integrin and ECM production are altered in physiological and pathological conditions such as memory, tumor, Alzheimer’s disease, stroke, and epilepsy (Clegg, et al., 2003; Dityatev & Fellin, 2008; Gall & Lynch, 2004). Although integrins are known to be involved in various physiological conditions and brain disorders, identifying the specific integrin involved and their precise role are difficult because many diseases are multifactorial and integrins are only one of these receptors involved. In this section, we will discuss recent reports on integrin role in the pathophysiology of learning and memory, and selected brain disorders.

3.1. Learning and Memory

Integrins are involved in learning and memory through modulation of long-term potentiation (LTP). The LTP has long been regarded as a plausible cellular substrate for learning and memory. At least two essential biochemical phases exist for LTP linked learning and memory: an early phase involving modification of existing synaptic proteins, and a late phase conveyed by de novo protein synthesis and formation of new synapses. It is thought that integrins help to form synapses in learning and memory. Integrins are involved in activity-dependent synaptic plasticity and in spatial memory. Evidence for a potential role in synaptic plasticity has been gathered by attenuating the stability of hippocampal LTP by using broad-spectrum peptide inhibitors of integrins or other pharmacological reagents. A direct link of integrin function to memory formation is demonstrated in Drosophila, in which the disruption of Volado, a gene encoding for two forms of the α-integrin, impairs short-term olfactory learning. Conditional expression of an integrin subunit rescued the memory deficits (Grotewiel et al., 1998).

Furthermore, disruption of the integrin-associated protein produces memory deficits in mice (Chang et al., 1999; Chang et al., 2001; Huang et al., 1998). Two vertebrate integrins of α8 and β8 have been localized to dendritic spines of pyramidal neurons where they are associated with the postsynaptic density (Einheber et al., 1996; Nishimura, et al., 1998). The α5 integrin has been shown to distribute preferentially to apical dendrites of pyramidal cells of the hippocampus and neocortex (Bi et al., 2001). Four different integrins of Drosophila have been localized to the presynaptic and/or postsynaptic side of the larval neuromuscular junction (Prokop, 1999). Heterozygous mutants of the integrin gene α3-subunit, but not of α5- or α8-subunit, reduce the magnitude of NMDA receptor dependent hippocampal LTP. However, when the expression of the three integrin genes, α3-, α5- and α8-subunits, is reduced simultaneously, a deficiency in spatial memory is produced but fear conditioning remains normal. These functional changes are associated with a fairly rapid but subtle morphological change in the structure of the synapse such that synaptic transmission is altered (Chan, et al., 2003).

Electrophysiological studies demonstrated that mutant or removal of β1-integrin have impaired synaptic transmission through AMPA receptors and diminished NMDA receptor-dependent LTP, and impaired in some kind of memories (Chan, et al., 2006; Huang et al., 2006). In addition, the β1-integrin receptors and their ECM ligands (FN, LN and CN) have been implicated in the process of neurite outgrowth and LTP (Chan, et al., 2003; Huang, et al., 2006; Venstrom & Reichardt, 1993). Neurite outgrowth is a process commonly thought to contribute to LTP by formation of new synaptic contacts that can be detected in the process of learning and memory. Activation of β1-integrin or several β1-binding α-integrins, including α3-, α5- and α8, further initiate intracellular signaling, and transcription and translation, and facilitate LTP related learning and memory.

Integrin ligands participate in the process of learning and memory. ECM molecule tenascin-C and a fragment of tenascin-C containing the fibronectin type-III repeats 6–8 are involved in hippocampus-dependent contextual memory and synaptic plasticity (Strekalova et al., 2002). The disruption of integrin-ECM interactions using RGD peptide and other integrin antagonists also interferes with the maintenance of use-dependent synaptic reorganization and LTP. Hippocampal synapses are enriched in FN receptors (i.e. α5β1 integrin) that contribute importantly to the stabilization of LTP (Bahr et al., 1997; Chun, et al., 2001; Kramar et al., 2002; Rohrbough et al., 2000; Staubli et al., 1998). Overall, integrins are essential for neuroplasticity in LTP and memory. Thus, future studies that elucidate the signaling cascade triggered by integrin engagement at the synapses will provide new insight into the processes of learning and memory.

3.2. Aging, Alzheimer’s disease and Down’s syndrome

Integrins may play roles in aging and age-related neurological disorders. Integrins are involved in synaptic plasticity in neurodegenerative conditions and immune response to the diseases. In humans, hippocampal pyramidal neurons and some neocortical neurons showed immunoreactivity with α4-integrin subunit and FN in all aged individuals, but not in younger patients. Antibodies against the α4-integrin subunit and a FN specific antibody also stained the tau-positive plaques (i.e. neuritic-type plaques) in Alzheimer’s disease and Down’s syndrome, while ‘preamyloid’ plaques remained negative (Van Gool et al., 1994). In addition, many senile plaques and neurofibrillary tangles in Alzheimer brain tissue are prominently stained for high level VN and VN receptor β3-integrin (Akiyama et al., 1991). The α4β1 and αLβ2 integrins in activated microglial cells adjacent to amyloidal plaques indicate inflammatory responses in Alzheimer disease (Preciado-Patt et al., 1996). In rat hippocampal neurons and rat cortical astrocytes, cell surface β-amyloid precursor protein is colocalized selectively with α1β1 and α5β1 integrins. In transfected human neuroblastoma cell line, α5β1 integrin appears to mediate the internalization and degradation of exogenous β-amyloid. When deposition of an insoluble amyloid around the α5β1-expressing cells is reduced, the cells show less apoptosis than the control cells (Matter et al., 1998; Yamazaki et al., 1997). Further studies on integrin such as α4β1 and α5β1 integrins in human brain tissue may provide more insight on the role of integrins in aging and Alzheimer’s disease.

3.3. Injury and Stroke

Integrins are believed to play an important role in neuronal injury and ischemic stroke. FN and α5β1 integrin are expressed at comparatively high levels in developing nerve, and less prominently expressed during nerve maturation. Following lesion of mature nerve or hippocampal hilus lesion by surgery, the up-regulated expression of FN and β1-integrin (e.g. α5β1 integrin) are in the vicinity of the lesion, in the growth cones of regenerating neurons and on Schwann cells (Lefcort et al., 1992; Pinkstaff et al., 1998). During peripheral neural repair and regeneration, activated integrins in microglia promotes adhesion, endocytosis, phagocytosis, and break down of cellular debris. Increased β1-integrin in neuronal cells relates to neuronal adhesion and neurite outreach and regeneration (Clegg, et al., 2003). Mature CNS neurons are believed lack the capacity to regeneration after injury except dentate granular cells (DGCs) in hippocampus. However, the regenerative performance of adult neurons can be restored to that of young neurons by gene transfer-mediated expression of a single α–integrin. Transfection of adult neuronal cells with α1-integrin results in increased neurite outgrowth on LN, while transfection with α5–integrin enhances neurite outgrowth on FN in vitro (Condic, 2001). Genetic variants with integrin α2-subunit have been reported to be associated with an increased risk for ischemic stroke (Matarin et al., 2008). After focal ischemic stroke, increased synthesis and release of matrix proteins osteopontin to the normal brain, and the increased expression of integrin αvβ3 indicated that osteopontin plays a novel role in glial activation, organization, and repair functions (Ellison et al., 1999). Following stroke, β1-integrin is increased in cerebral blood vessel in ipsilateral ischemic cortex, and is involved in modulating angiogenesis in ischemic stroke (Lathia et al., 2010).

In both rat and mouse stroke models, western blot analysis revealed elevated levels of ECM fragment of perlecan (domain V). Post-stroke domain V administration increased vascular endothelial growth factor levels through brain endothelial cell α5β1 integrin, and the subsequent neuroprotective and angiogenic actions of perlecan domain V were in turn mediated through vascular endothelial growth factor receptors (Lee et al., 2011). Becker et al reported that treatment with α4-integrin Ab decreased infarct size, increased lymphocyte/monocyte and lowered neurological deficit scores in rat focal cerebral ischemia model (Becker et al., 2001). These results suggest that integrins could represent a promising approach for stroke treatment.

3.4. Epilepsy

There is emerging evidence on the role of integrins in epilepsy. Epilepsy is the second most common neurological disorder that affects over 3 million people in the US and about 50 millions worldwide (Browne & Holmes, 2001). It is a disorder in which the balance between neuronal excitability and inhibition is disrupted, leaning toward uncontrolled synchronized excitability. In temporal lobe epilepsy (TLE), the hippocampus plays a key role in epileptogenesis and epileptic seizures (Reddy, 2010; Schwartzkroin, 1994). Aberrant expressions of neurotransmitter receptors, cytoskeletal proteins, synaptic proteins, antioxidant proteins, and MEK have been found in the hippocampus of patients with TLE (Furtinger et al., 2003; Notenboom et al., 2006; Perosa et al., 2007; Yang et al., 2006). In addition to these intracellular modifications, there is increasing evidence for a functional correlation between the localization of integrins in the hippocampus and the role that integrins may play in neuronal epileptiform activities (Chang et al., 1993; Grooms & Jones, 1997).

Integrins are differently distributed in cellular and subcellular compartments in the hippocampus regions and undergo specific patterns of regulation during development of epilepsy. Immunocytochemical and co-precipitation studies have suggested 11 potential integrin receptors with species difference in the hippocampus including α1β1, α2β1, α3β1, α4β1, α5β1, α6β1, α8β1, αvβ1, αvβ3, αvβ5, αvβ8 (Chan, et al., 2003; Chun, et al., 2001; Fasen, et al., 2003; Pinkstaff, et al., 1999). Significant hippocampal immunoreactivity of α1-, α5-, β1-, β3-, β4-, and β5-integrins is observed in the pia mater, in vascular endothelia, and in astrocytes at the pial surface; immunoreactivity of β2-integrin is found exclusively in vascular endothelia, and immunoreactivity of α2-, β4-, and β5-integrins is found in mossy fibers. Pyramidal cells, interneurons of CA1–CA3, and hilar neurons reveal moderated α5-integrin labeling in their cell bodies. After pilocarpine-induced status epilepticus, strong immunoreactivity for α1-, α2-, α4-, α5-, β1-, β3-, and β4-integrins is observed in reactive astrocytes (Fasen, et al., 2003). Seizures induce marked increases in αv- and α1-integrins that are restricted to neuronal cell layers and are particularly striking in hippocampal stratum granulosum, CA1 and CA3.

Our immunocytochemistry data show at least 20% increase in α5-integrin expression in hippocampal DGCs in stage 5 kindling mice (unpublished data). In contrast, seizure-induced increases in α6-integrin are more diffusely distributed and are localized in both neurons and glia (Gall & Lynch, 2004). The β1-integrin expression is reported broadly increased and have been implicated in the process of neurite outgrowth during the seizure episode (Gall & Lynch, 2004; Venstrom & Reichardt, 1993). Hippocampal neurons and astroglial cells express quite low levels of β1–integrin mRNA in naive rats. However, β1-integrin expression can be dramatically increased in response to seizure within hippocampal stratum pyramidale and the dentate gyrus hilus. β1-integrin mRNA levels increased first in neurons and peaked with predominant astroglial expression at 24 h after seizure (Pinkstaff, et al., 1998). Laminin β1-subunit and integrin α2-subunit expression are also elevated in the anterior temporal neocortex tissue from patients with intractable epilepsy (Wu et al., 2011b).

Integrins as unique candidates may participate in epileptogenesis. There is increasing evidence for a functional correlation between the localization of integrins in the hippocampus and the role that integrins may play in neuronal epileptiform activities (Chang, et al., 1993; Grooms & Jones, 1997). Seizure activity has been shown to induce a ‘matrix response’. These responses included release of protease, changes in the localization and synthesis of ECM molecules (e.g. FN, reelin, and glycosaminoglycans), and activation of integrin-intracellular signaling pathways following seizure activity in the hippocampus (Gong et al., 2007; Hoffman et al., 1998b; Hoffman et al., 1998c; Naffah-Mazzacoratti et al., 1999; Perosa et al., 2002a; Perosa et al., 2002b). In animal epilepsy and in human TLE, neurons in hippocampus undergo extensive remodeling, including acute neurodegeneration and intracellular signaling adjustments, and chronic processes including reorganization of mossy fibers, dispersion of the DGC layer and the appearance in ectopic locations within the dentate gyrus. Integrin recruitment and relocalization has been observed in migrating cells, and integrin-ECM attachments and focal adhesions have been reported to dynamically change their position at leading and rear edges (Becchetti & Arcangeli, 2010; Huttenlocher & Horwitz, 2011). Our data show decreased FN-integrin binding force/adhesion force and increase cell membrane stiffness/plasticity in DGCs from stage 5 kindling mice by using nanoscale atomic force microscopy (unpublished data). These results indicate alternation of ECM-integrin interaction represented as ECM-integrin binding changes and alternation of intracellular CSK remodeling and intracellular molecules activation represented as stiffness/plasticity changes during epilepsy.

As we mentioned above, epilepsy is a disorder in which the balance between neuronal excitability (e.g. NMDA currents) and inhibition (e.g. GABA currents) is leaned toward uncontrolled excitability. Integrin expression is greatest in glutamatergic (NMDA) neurons and low in GABAergic neurons and glia, and concentrated in specific areas such as synaptic membrane (Gall & Lynch, 2004). There are several reports regarding β1- integrin family activated excitatory NMDA currents and Ca²⁺ currents in neurons as discussed above (Bernard-Trifilo, et al., 2005; Gui, et al., 2006; Lin, et al., 2003; Lin et al., 2008; Lin et al., 2005). All of these changes will increase glutamate excitability and burst activities in hippocampal CA1 and DGC, cause hyperexcitation in neurons, and could be primary events leading to development of the epilepsy (Bernard-Trifilo, et al., 2005; Chang, et al., 1993; de Lanerolle et al., 1989; Gui, et al., 2006; Lin, et al., 2003; Lin, et al., 2008; Lin, et al., 2005; Smith & Dudek, 2002; Sutula et al., 1988; Tauck & Nadler, 1985; Wuarin & Dudek, 2001). In epileptic conditions, increased neuronal proliferation in the DGCs which principal excitatory glutamatergic phenotype neurons, cause hypersynchronization (Kokaia, 2011). However, there are still limited reports available on whether or how integrin directly or indirectly modulates GABA channels. Colocalization of α3-integrin subunit and the GABA_A receptor β2/3 subunit has been reported in the Purkinje neuron. Activating α3β1 integrin impairs the GABAergic miniature inhibitory postsynaptic currents (Kawaguchi & Hirano, 2006). ECM protein tenascin-R-deficient mutant mice show impaired LTP, elevated levels of excitatory synaptic transmission, and reduced levels of perisomatic inhibitory currents mediated by GABA_A receptors in CA1 of the hippocampus (Bukalo et al., 2007). These reports reveal the complex interplay between integrins and GABAergic neurotransmitter receptors. Because glutamatergic activities related to LTP as mentioned above, these results indicate that integrin contributions to plasticity are not restricted to ‘good things’ as LTP to memory, but included ‘bad things’ as long lasting increases in excitatory LTP, strengthen the network and connectivity, and lower the threshold for epileptogenesis.

Epileptogenesis involves a local breakdown of normal cell-cell and cell-matrix relationships followed by new synthesis of ECM and integrins. During normal neural development, ECM molecules play an important role in neuroplasticity events such as neurogenesis, axonal outgrowth, and synaptogenesis. Synaptogenesis is associated with the early stages of epilepsy formation and these plasticity changes are also involved in the development of epilepsy (Chang, et al., 1993). ECM-integrin interactions may alter neuronal signaling through the RGD binding site or increasing the number of activated integrins in epilepsy (Grooms & Jones, 1997; Morini & Becchetti, 2010). The RGD-treated brain slices display a significant decrease in the spontaneous burst rate (epileptic-like activities), but the period of spontaneous bursting increase dramatically in the hippocampus CA3. This paradoxical effect might be because RGD peptide decreases the number of available integrin-RGD containing ECM protein binding sites, which may interfere with neuronal communications required for synchronized electrical firing rates (Grooms & Jones, 1997). Kainic acid–induced seizure caused a broadly distributed increase of FN as early as 1 hour after recurrent seizure in adult brain hippocampus including dentate gyrus hilus and cortex as well as a larger focal increase in the ventral subiculum. FN density is higher in cell hillock within individual cells in dentate gyrus hilus (Hoffman, et al., 1998c). The hippocampus of TLE patients show increased levels of chondroitin sulfate and hyaluronic acid, another two components of ECM protein glycosaminoglycans. The increased concentration of hyaluronic acid and chondroitin sulfate in the hippocampus of TLE patients demonstrates the importance of the matrix compounds during neosynaptogenesis, neurite outgrowth, and the mossy fiber sprouting found in temporal lobe epilepsy phenomena (Perosa, et al., 2002a).

The expression of ECM molecule tenascin-C, which could binds to α8β1, α9β1, αvβ3 and αvβ6 in nervous system (Clegg, et al., 2003; Nakic et al., 1996; Shapiro et al., 1996; Varnum-Finney et al., 1995), reaches peak upregulation at 24 hours after kainic acid-induced limbic seizures in the CA1 and granule cell layer of adult rat, coincident with activation of granule cells and sprouting of axon terminals. The remaining tenascin expression 30 days after injection only relates to pyramidal cells in CA1 and CA3, coincident with an astroglial response (Nakic, et al., 1996). During status epilepticus, overall ECM protein hevin SC1 levels decrease in DGC. A transient increase in SC1 co-localization with the cellular stress marker Hsp70, the degeneration marker Fluoro-Jade B, and the neuron activity marker activity-regulated CSK-associated protein are also shown in neurons of the hippocampal CA1, CA3, and hilar regions after status epilepticus. The levels of SC1 protein in neurons of the hippocampal seizure-resistant CA2 region do not change throughout the seizure (Lively & Brown, 2008).

α-Tubulin, a component of ECM-integrin–FAC (or CSK) axis, exhibits increased expression in CA3 and DGC bodies, dendrites and axons of hippocampus in epilepsy models. Microtubule formation may contribute to synaptic remodeling, such as mossy fiber sprouting and reorganization of neural networks of kindling-induced epileptogenesis (Hendriksen et al., 2001; Pollard et al., 1994; Sato & Abe, 2001). However, in mesial TLE patients, tubulin α-1 chain, β-tubulin, profilin II, and neuronal tropomodulin are significantly reduced, whereas ezrin and vinculin are significantly increased (Yang, et al., 2006). Divergent results from animal and patients may be explained by methodological differences, timing these molecules is examined in animal and patients, as well as discrepancies between gene and protein levels. Overall, these reports suggest that ECM-integrins-CSK is involved in the pathophysiology of epilepsy.

Currently available treatments of epilepsy are only seizure suppressing ‘antiepileptic’; none are truly disease modifying ‘anti-epileptogenic’ treatment. The major goal of future epilepsy research is to identify related underlying biological pathways and mechanisms responsible for epileptogeneis, and drug resistance in certain epilepsy patients. Integrin neurobiology may provide novel target to develop new drug candidates as ‘anti-epileptogenic’ (Pack et al., 2011; Reddy, 2010).

4. Therapeutic Implications of Integrins as Drug Targets

Integrins are being explored for designing drugs for the treatment of neurological disorders. Because successful axonal regeneration of attachment, migration, and extension processes are highly correlated with the induction of integrins on the surface of neurons, peptides or other types of ligands derived from ECM proteins may have therapeutic potential for treatment of brain diseases (Meiners & Mercado, 2003).

4.1. Approaches to integrin drugs

Rapid progress has been made in the discovery and development of integrin targeted therapeutics in the past 15 years. The first integrin-specific drugs target the platelet integrin, αIIbβ3 and have been marketed for acute coronary syndromes and prevention of myocardial infarction following percutaneous coronary intervention since the late of 1990s. This drug has been proven to be effective and safe and contributed to the reduction of mortality and morbidity in acute coronary syndromes. There are numerous in vitro, in vivo, preclinical and clinical studies implementing integrins as potential drug candidates for human diseases (Table 2). There are several mechanisms to modulate integrin function for drug development. The first mechanism as the original strategy for currently approved inhibitors is to block the ligand binding (i.e. block adhesion). Integrin inhibitors targeted on three integrins of αIIbβ3, α4 and αLβ2 has been approved. Currently, integrin Abs, integrin ligand peptides and small non-peptide inhibitors have been clinically targeted. Abs can have longer circulation lifetimes compared to small peptide-based and non-peptide drugs thus increasing the duration of therapy. However, the disadvantages associated with antibody therapeutics are the high cost of production, the need for intravenous administration, and the propensity for host immunogenicity and infusion reactions (Cox, et al., 2010; Millard, et al., 2011).

Table 2. Integrin as target of drugs.

Data compiled from: (Clemetson & Clemetson, 1998; Horwitz, 1997; Millard, et al., 2011; Ross & Borg, 2001; Staunton et al., 2006; Wu & Davis, 1998)

Integrin	Cell types	Integrin ligands	Example of disorder indication and drugs
αIIbβ3* (CD41, GPIIbIIIa)	Platelets	Fibrinogen, von Willebrand factor, FN, VN	Acute coronary syndrome*, MI, restenosis after angioplasty, percutaneous coronary intervention*, thrombosis. Drugs: Abciximab, Epifibatide, Abeiximab, etc
β2 integrins* (CD18)	Various white blood cells	ICAM	Reperfusion injury, stroke, shock, MI, multiple sclerosis, psoriasis*, chronic inflammatory diseases, meiningitis Drugs: Erlizumab, Efalizumab (αLβ2)#, etc
β1 integrins (CD29)	Widespread including nervous system	FN, LN, CN, others	Platelet aggregation, angiogenesis, homeostasis, wound healing processing, hypertrophy Drugs: see individual αβ-integrin.
α4β1* (CD49d)	Various white blood cells, nervous system	FN, VCAM	Multiple sclerosis*, chronic inflammatory diseases, such as Crohn’s disease*, asthma, and arthritis Drugs: Tysabri, Natalizumabetc
α5β1 (CD49e)	Smooth muscle cells, cardiomyocytes, nervous system	FN (possible biomarker for infraction)	Restenosis, muscle dystrophy, tumors, age- related macular degeneration Drugs: Volociximab, etc
αvβ3 (CD51)	Endothelial cells, vascular smooth muscle	FN, VN, others	Angiogenesis, glioblastoma, diabetic retinopathy, heart defect, atherosclerosis, prostate cancer, pancreatic cancer, melanoma and postmenopausal osteoporosis Drugs: Cilengitide, Vitaxin, MK0429, etc
α4β7	Various white blood cells	FN, MAdCAM, VCAM	chronic inflammatory diseases, such as ulcerative colitis Drugs: MLN02

FN: fibronectin; VN: vitronectin; CN: collagen; LN: laminin; MAdCAM: mucosal addressin cell adhesion molecule; VCAM: vascular cell adhesion molecule; ICAM: intercellular adhesion molecule; MI: myocardial infarction.

^*FDA approved integrins that can be used in diseases with bold font.

^#withdrawn from market in 2009.

The second mechanism is to modulate integrin expression and activation. As recent advances in our understanding of ECM-integrin-intracellular signaling axis, blocking downstream integrin signaling for both outside-in and inside-out will modulate integrin expression and activation. For instance, CSK talin binding, known forming the initial contacts between integrin β-subunit intracellular tails and the actin cytoskeleton, is a pivotal event and is required specifically for integrin-ECM-CSK axis signaling and for activation of integrin (Kim, et al., 2011; Millard, et al., 2011). If talin binding to intracellular β-integrin tail is inhibited, it will prevent inappropriate integrin activation and block subsequent integrin signaling. Similarly, NRTKs such as FAK and Src binding to β-tails and CSK protein paxillin binding to α-subunit intracellular tails represent additional potential key targets for development of selective inhibitors in ECM-integrin-FAC pathways. Some inhibitors such as HYD1 for α6β1 and ATN-161 for FN sequence to block signaling have been studied in clinical or pre-clinical trials (Cianfrocca et al., 2006; Sroka et al., 2006).

4.2. Potential clinical applications

Integrin targeted agents may have clinical applications in brain disorders. Integrins regulate the transit of lymphocytes, macrophage, and other cells across the blood–brain barrier in response to inflammatory stimuli, anti-integrin reagents are of great interest as therapeutic agents to control demyelinating diseases, such as multiple sclerosis that involve the immune system and inflammatory responses. A new potential approach that complements traditional therapy, targeted to specific integrin receptors using integrin ligands, has been reported. The anti-α4-intergin Ab natalizumab, which inhibited attachment of immune-competent cells (leukocyte) to inflamed brain endothelium, demonstrated an unequivocal therapeutic effect in preventing relapses and slowing down the pace of neurological deterioration in patients with relapsing-remitting multiple sclerosis. Combinational treatment with both anti-β7 and α4 integrin subunit Abs led to more rapid and complete remission than that obtained with anti-α4 antibody alone. These reports concluded that α4β1, α4β7, and αEβ7 integrins may all play a contributory role in the progression of chronic forms of demyelinating disease, and together with their ligands could represent potential targets for improved treatment of some forms of multiple sclerosis (Bartt, 2006, Kanwar, 2000 #1809; Engelhardt & Kappos, 2008; Kanwar et al., 2000). Treatment with natalizumab (α4-integrin) also led to a dramatic reduction of refractory seizures in a patient with multiple sclerosis (Sotgiu et al., 2010). The involvement of integrins in inflammatory responses has also created interest in the possibility of using anti-integrin reagents to alleviate several neurodegenerative disorders, including Parkinson’s and Alzheimer’s diseases. Of special interest, α1β1, α6β1 integrins and CD47 (integrin-associated protein) appear to interact with amyloid precursor protein and these are postulated to mediate deposition or toxic actions of A and amyloid formation (Bozzo et al., 2004, Koenigsknecht, 2004 #1812; Koenigsknecht & Landreth, 2004).

Integrin antagonists that target angiogenesis for brain tumors are progressing through clinical trials. Cilengitide, a selective inhibitor of αvβ3 and αvβ5 integrins, inhibits cell signaling through focal adhesion kinase/Src/Akt and extracellular signal-regulated protein kinases mediated pathways, and attenuates the effect of vascular endothelial growth factor stimulation on growth factor signaling in endothelial and glioma tumor cells. Following cilengitide treatment, endothelial and glioma cells show disassembly of cytoskeleton and disruption of tight junction formation (Oliveira-Ferrer et al., 2008). The addition of cilengitide to chemoradiotherapy based treatment regimens has shown promising preliminary results in ongoing clinical trials in newly diagnosed and progressive glioblastoma multiforme patients by inhibiting angiogenesis (Millard, et al., 2011). The greatest challenge facing the development of anti-angiogenic integrin targeted therapies is the overall lack of biomarkers by which to measure treatment efficacy.

4.3. Drug delivery in nervous system

The major problem in drug delivery to CNS is the presences of the blood-brain barrier (BBB) and blood cerebrospinal fluid barrier, the mechanisms that protect CNS against intrusive chemicals also confound therapeutic interventions. Compare to BBB, blood cerebrospinal fluid barrier allows the free-movement of small molecules. Many existing drugs including currently clinical available integrin drugs are rendered ineffective in the treatment of many CNS diseases due to our inability to effectively safely deliver, and sustain them within the CNS. To circumvent the barriers, at least three strategies are practically possible and have been evaluated. There are manipulating drugs, disrupting the BBB and discovering alternative routes for drug delivery (Misra et al., 2003). Designer drugs are prepared through increasing the lipophilicity of therapeutic drugs, and vector (e.g. nanoparticles) or receptor and carrier (e.g. levodopa) mediated drug delivery. Disrupting the BBB is carried out through hypertonic solution initiating endothelial cell shrinkage and vasoactive leukotriene increasing cell permeability. These two strategies delivered via circulatory system and frequently results unwanted side effects. The third strategy is to bypass BBB and deliver the drugs to CNS through intraventricular/intrathecal route, olfactory pathway, interstitial/intracranial drugs delivery via releasing drugs from biological tissues, nanoparticles and pumps.

Nanoparticles as a drug-carrier system for the antiepileptic valproic acid have been studied in mice. This study suggested that nanoparticles loaded with valproic acid may help to reduce the toxic side effects of valproate therapy, not by reducing the therapeutically necessary dosage but by inhibition of formation of toxic metabolites (Darius et al., 2000). Wilson et al. have used Poly-nanoparticles for the targeted delivery of rivastigmine into the rat brain to treat Alzheimer’s disease (Wilson et al., 2008). Targeted gene therapy with αvβ3 integrin-binding nanoparticles as treatment for choroidal neovascularization has also been reported (Salehi-Had et al., 2011). Sniffing (intranasal) neuropeptides of melanocortin, vasopressin and insulin achieved direct access to the cerebrospinal fluid within 30 minutes, bypassing the bloodstream in human trials (Born et al., 2002). RGD peptides are small peptides that could be applied through this pathway. In addition, application of multifunctional nanocarriers with integrin ligands across the BBB, allowing for diagnostic and therapeutic agents to be selectively targeted on a specific tissue and cell, also minimizing exposure to the healthy tissue and cell, is one of enormous challenges for biological discovery and clinical practice.

BBB dysfunction in the hippocampus could be present in a chronic hypertensive state at an early stage, aged senescence-accelerated mice, and during inflammatory reaction. BBB structure could be compromised during stroke, tumor metastasis, and injury (Cox, et al., 2010; Pelegri et al., 2007; Ueno et al., 2004). These could provide an opportunity for applying anti-integrin drugs to CNS.

The road from basic research to clinical application of integrin pharmacology has been very challenging. Approximately 2–4% of patients had anaphylaxis or other hypersensitivity reactions to natalizumab. Combined natalizumab and interferon-β1a therapy developed progressive multifocal leukoencephalopathy in multiple sclerosis because of their immune suppressive properties (Engelhardt & Kappos, 2008; Warnke et al., 2010). Thus Natalizumab and related α4-integrin targeting drugs come with a black-box warning on the drug label and are now limited to patients refractory to standard therapies. In clinical practice, physicians face the tough challenge of accurately evaluating both the benefits and the risks of therapy for the patients. The real challenges are that there are lacks of fully understanding of the function of integrins and the role of integrins in diseases, in particular their roles in signaling.

5. Conclusions and Future Perspectives

In conclusion, ECM-integrin-CSK interactions are involved in rearrangements of cell body, axonal, dendritic, and astrocytic processes. However, the extent of integrin regulation of the synaptic events during neuronal diseases remains unclear. ECM-integrin-CSK-intracellular signaling interactions in nervous system could play a major role in the responses of neurons and blood vessels to injury and repair. Both neuronal activity and vascular reactivity from tissue injury rapidly modulate integrin signaling with consequent modulation of ion channels and activities, the generation or exposure of new integrin ligands from limited degradation of extracellular matrix through increased extracellular proteases, and/or turnover of new integrins (Banes et al., 1995; Bernard-Trifilo, et al., 2005; Davis, et al., 2000; Davis, et al., 2002; Endo et al., 1999; Fukai et al., 1995; Gualandris et al., 1996; Gui, et al., 2006; Hoffman, et al., 1998a; Hoffman, et al., 1998b; Jones et al., 1995; Kramar, et al., 2003; Lin, et al., 2003; Martinez-Lemus, et al., 2003; Momota et al., 1998; Waitkus-Edwards, et al., 2002; Wu, et al., 1998; Wu, et al., 2008b; Wu et al., 2000; Yang, et al., 2010). One of the further studies are warranted to study how integrins modulate fast dynamic changes of ion channels such as GABAergic inhibition.

Integrins as receptors are attractive targets for pharmacological manipulation of integrin-based signaling. Specificity or selectivity is a challenge to develop therapies against integrins. To avoid or lower risk of side effects, drugs need to distinguish between integrins expressed in healthy and injured tissues, target to activated integrin receptor not only resting integrins, and compete for single αβ combinations, not only inhibiting discrete α or β subunits. For instance, αvβ3 and αIIbβ3 share β3 subunit and both recognize the RGD motif; drugs that inhibit β3 subunit as αvβ3 antagonist to inhibit angiogenesis will lead to side effects of bleeding from inhibiting αIIbβ3. Advances in structural characterization of integrin-ligand interactions will prove and has proved beneficial in the design and development of potent and selective inhibitors for each single combination. The small molecule antagonists that are small enough to block ligands bind to α–subunit which determines ligand specificity, without interacting with β-subunit which activates signaling pathway, are also potential strategy to achieve.

In neurological diseases, ECM proteins often degrade or denature. Development of integrin ligands that recognize these degraded or newly expressed ECM components is critical. In addition, it is not clear whether degraded integrin ligands serve as early biomarkers for diagnostic purposes. Recent reports support the utility of integrin ligands as potential biomarkers for diagnosis and treatment of tumor and cardiovascular diseases (Cai et al., 2008; Martin-Ventura et al., 2009). α6β4 integrin to laminin of Schwann cells and myelin is detected in the serum of Guillain-Barre syndrome patients while control is negative. Therefore, circulating α6β4 may provide a marker for extensive peripheral myelin damage and may be used to validate clinical diagnosis of Guillain-Barre syndrome, evaluate its course and activity (Sessa et al., 1997). Future studies on integrin structures and functions in the normal and diseased brain will provide interesting insights and open doors for the development of both allosteric and activation-specific drugs. Overall, integrin targeted drugs may hold promise for treatment of specific neurological conditions including epilepsy and Alzheimer’s disease.

Abbreviations

Ab

antibody

AMPA

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

BBB

blood-brain barrier

Ca_L

L-type Ca²⁺ channels

CN

collagen

CNS

central nervous system

CSK

cytoskeleton

DGC

dentate granular cell

ECM

extracellular matrix

EGF

epidermal growth factor

EPSP

excitatory postsynaptic potentials

ERK

extracellular signal-regulated protein kinases

FAC

focal adhesion complex

FAK

focal adhesion kinase

FN

fibronectin

GABA

γ-aminobutyric acid-A

LDV

leucine-aspartate-valine

LN

laminin

LTP

long-term potentiation

MEK

mitogen-activated protein kinase

NDMA

N-methyl-D-aspartic acid

NRTK

non-receptor tyrosine kinase

PNS

peripheral nervous system

RGD

arginine-glycine-aspartate

TLE

temporal lobe epilepsy

VN

vitronectin