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(Mandell et al., 2001). Activation of ERK/MAPK pathway has been shown in ONH astrocytes in vitro and in vivo in glaucoma and in astrocytes exposed to hydrostatic pressure (Tezel et al., 2003; Hashimoto et al., 2005).

G protein-coupled receptors

Signaling through G protein-coupled receptors (GPCRs) mediates numerous cellular functions including mechanotransduction (Chachisvilis et al., 2006). The effects of activation of G proteincoupled metabotropic glutamate receptors, GABA receptors, nucleotides, and catecholamines in brain astrocytes are under intense study. Endothelin-1 (ET-1) has been proposed to play role in astrogliosis that occurs in the glaucomatous ONH. G protein-coupled endothelin-1 receptors ETA and ETB are abundantly expressed and widely distributed in ocular tissues, including the retina, optic nerve, and ONH astrocytes as described recently (Prasanna et al., 2002; Chauhan et al., 2004; Wang et al., 2006). In the CNS, ET-1 regulates astrocyte cell shape through tyrosine phosphorylation of FAK and paxillin (Koyama et al., 2000). Recently, it was shown that the endothelin receptor B is expressed in ONH astrocytes in human glaucoma (Wang et al., 2006). ONH astrocytes and lamina cribrosa cells express functional endothelin receptor A and endothelin receptor B (ETB) in culture (Rao et al., 2007). Moreover, ET-1 regulates the expression of matrix metalloproteinases (MMPs) and tissue inhibitors in ONH astrocytes further suggesting that the ET pathway may be involved in tissue remodeling in glaucoma (He et al., 2007).

The regulators of G protein signaling (RGS) are an important way to modulate signal transduction (RGS5 ocular). RGS5 protein and mRNA are abundantly expressed in human ONH astrocytes in vivo and in vitro (Fig. 5). Current studies suggest that the function of RGS5 is to modulate astrocyte responses in response to pressure. Among the known functions of RGS5 the modulation of G protein-coupled receptors such as prostaglandin EP3 and EP4 which affect the transcription of genes such as NOS2 (Hu et al., 2005). Figure 5 illustrates preferential localization of RGS5 to laminar astrocytes in the glaucomatous human

ONH of Caucasian donors compared to normal donors (Fig. 5B) compared with low levels of RGS5 in astrocytes in the normal ONH (Fig. 5A). Consistent with the immunohistochemistry, primary cultures of ONH astrocytes from glaucomatous donors exhibit higher levels of RGS5 protein compared with normal donors by Western blot analysis (Fig. 5C). Real time quantitative RT-PCR detected significant higher mRNA levels for RGS5 in primary cultures of ONH astrocytes from Caucasian donors with POAG compared to normal donors (Fig. 5D).

Ras superfamily of small G proteins

The Ras superfamily members participate in many cellular processes including cell movement and act as signal transducers and/or regulators of membrane trafficking (Seachrist and Ferguson, 2003). Ras proteins cycle between a GTP-bound state and a GDP-bound state because of their GTP hydrolysis activity and a higher affinity for GTP than GDP. In vivo, this cycle is regulated by GTPase activator proteins, which increase the rate of GTP hydrolysis, and guanine nucleotide exchange factors, which stimulate the exchange of GDP for GTP. Members of the Rho, Rab, and Ran families are also regulated by GDP dissociation inhibitors, which, by binding to the GDP-bound form, inhibit nucleotide exchange. Rac1 and Cdc42 regulate migration in astrocytes because of their ability to regulate actin cytoskeletal dynamics by signaling through effectors of the Ras-activated kinases (Wheeler et al., 2006; Bourguignon et al., 2007). CDC42, a small Rho GTPase that participate in polarized motility in astrocytes (Osmani et al., 2006) was upregulated in ONH astrocytes exposed to hydrostatic pressure at the mRNA and protein levels. We also demonstrated early increased Rho activity in ONH astrocytes exposed to hydrostatic pressure (Yang et al., 2004).

Astrocyte migration in the glaucomatous optic nerve head

Migration of astrocytes occurs during normal development, in neurodegenerative diseases, after

injury, and during tumor invasion in the CNS. Cell migration is a biochemical and mechanical response that can originate via in response to ECM molecules such as laminin, fibronectin, or environmental cues such as growth factors or cytokines including the TGFb family (Yu et al., 2007), and EGF (Liu and Neufeld, 2007). Most importantly, in glaucoma, reactive astrocytes have been shown to migrate from the cribriform plates into the nerve bundles (Liu and Neufeld, 2004) and synthesize neurotoxic mediators such as nitric oxide (NO) and TNF-a, which may be released near axons causing neuronal damage (Liu and Neufeld, 2000; Neufeld and Liu, 2003; Tezel et al.,

2004). These findings suggest that migration of astrocytes likely precedes the remodeling in the optic neuropathy.

Exposure of ONH astrocytes to elevated hydrostatic pressure in vitro increased cell migration (Fig. 6E, F) through activation of at least two signaling pathways: PI-3K activation and pressure induced erbB2, EGFR1, and PDGFR kinases activation. Interestingly, inhibition of tyrosine kinase receptors did not affect astrocyte basal migration induced by the cell-free area. On the other hand, mechanical denudation induced migration by a rapid and sustained increase in COX2 activity (Salvador-Silva et al., 2004). The

importance of these findings is that in a model of reactive astrocytes in vitro, separate mechanisms govern migration in response to mechanical injury and in response to a physical stress such as elevated pressure.

Previous work in our laboratory demonstrated changes in astrocytes cytoskeleton after exposure to hydrostatic pressure in the intact monolayer (Wax et al., 2000; Salvador-Silva et al., 2001). These changes included redistribution of actin to the cell margins and localization of GFAP and vimentin intermediate filaments to the perinuclear region. In these experiments, astrocytes in the intact monolayer appeared flat and polygonal in shape when exposed to control pressure. GFAP filaments were organized as a diffuse network running radially toward the edges of the cell border (Fig. 6C). In astrocytes migrating into the cell-free area under ambient pressure, GFAP (Fig. 6A) staining was diffuse but evenly distributed throughout the cell. Astrocytes in the intact monolayer exposed to HP appeared elongated in response to HP (Fig. 6D). GFAP staining was stronger around the nuclear region. In migrating astrocytes exposed to HP, GFAP (Fig. 6B) staining was strong around the nucleus towards the rear of the cell.

Cell adhesion of ONH astrocytes

Reactive astrocytes express several cell adhesion molecules including cadherins, integrins, and neural cell adhesion molecules (NCAM) in CNS

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degenerations (Charles et al., 2000). Three NCAM isoforms result from alternative splicing of a single gene: two major transmembrane isoforms,

denoted NCAM140 and NCAM180, and NCAM120 a glycosyl-phosphatidylinositol-linked isoform (Maness and Schachner, 2007). The adhesive properties of NCAM depend on alternative splicing of the primary transcript or post-translational modifications, such as polysialylation (Maness and Schachner, 2007). Activation by extracellular cues of cellular signaling pathways regulates alternative splicing. Recent studies suggest that c-Jun, a transcription factor, regulates alternative splicing of NCAM pre-mRNA and the synthesis of NCAM140 (Feng et al., 2002). In the adult human ONH, quiescent astrocytes and lamina cribrosa cells express NCAM140 as the predominant isoform (Ricard et al., 2000) NCAM120 and NCAM180 are not expressed by quiescent ONH astrocytes in vivo or in vitro (Ricard et al., 2000; Hernandez et al., 2002). In the human glaucomatous ONH, reactive astrocytes differentially express NCAM180 mRNA and protein (Hernandez et al., 2002). Injury to the mouse optic nerve increases expression of NCAM180 in the axons and in reactive astrocytes (Becker et al., 2001).

Integrin receptors cluster at focal adhesion complexes, where cells anchor into the ECM. Binding at these sites can activate intracellular signaling pathways to modify cell behavior, including cell migration, differentiation, adhesion to substrates, and target recognition. Integrins a2a1, a3ab1, a6b1, and a6b4 provide attachment for ONH astrocytes to basement membranes via