(Dawson and Dawson, 1996; Lipton, 1999). Additionally, peroxynitrite can elicit Zn2+ release from intracellular stores, resulting in loss of mitochondria membrane potential and subsequent neuronal death (Bossy-Wetzel et al., 2004). In the rat retina, intravitreal injection of NMDA increased formation of peroxynitrite, and NMDA-induced RGC death was inhibited by pharmacological inhibition of peroxynitrite formation (El-Remessy et al., 2003). Pathological activation of poly(ADP-ribose) polymerase-1 (PARP-1) after DNA damage is also thought to contribute to cell death by depleting cellular ATP, among other mechanisms, including AIF release (Dawson and Dawson, 1996; Yu et al., 2006). PARP-1 activation occurs in the retina after NMDA injection in the vitreous and is accompanied by a decrease in ATP levels (Goebel and Winkler, 2006). Conversely, a PARP inhibitor protects RGCs from NMDA-induced excitotoxicity (Goebel and Winkler, 2006).

An additional pathway in NMDA excitotoxicity involves p38 mitogen-activated protein kinase (MAPK), which is activated through phosphorylation of its serine and threonine residues. Although initial findings associated p38 MAPK with inflammatory responses (Lee et al., 1994), later studies showed that activation of p38 MAPK can be involved in apoptosis (Xia et al., 1995). Our group has shown that activation of p38 MAPK also occurs in the retina after intravitreal injection of NMDA (Manabe and Lipton, 2003). Moreover, specific p38 MAPK inhibitors can ameliorate NMDA-induced excitotoxicity (Kawasaki et al., 1997; Manabe and Lipton, 2003). A recent study identified Rho GTPase as an essential molecule that links elevation of [Ca2+]i to p38 MAPK activation and subsequent excitotoxic neuronal death (Semenova et al., 2007). NO or ONOO is also capable of activating p38 MAPK upon excitotoxic stimuli (Bossy-Wetzel et al., 2004; Cao et al., 2005). In contrast, depending on the circumstances, p38 MAPK can also trigger a survival-promoting pathway through activation of the downstream transcription factor myocyte enhancer factor 2C (MEF2C) (Mao et al., 1999; Okamoto et al., 2000).

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The degree of excitotoxic insult will influence a neuron to undergo apoptosis or necrosis. Exposure to a low concentration of NMDA induces neuronal cell death with apoptotic morphology, whereas a high concentration of NMDA induces predominantly necrosis (Ankarcrona et al., 1995; Bonfoco et al., 1995). The duration of insult is also important. We thus hypothesized that chronic glutamate receptor hyperactivity, even if mild, could trigger apoptotic cell death, possibly following initial synaptic damage (Lipton, 2004). In support of this premise, we and our colleagues reported that mild but prolonged elevation of glutamate levels in the vitreous (30 mM for 3 months, with normal concentrations B13 mM) resulted in loss of approximately 40% of rat RGCs (Vorwerk et al., 1996). This form of slow and subtle excitotoxicity, leading to oxidative (ROS-related) and nitrosative (NO-related) stress, has been implicated in a variety of chronically progressing neurodegenerative disorders, possibly including glaucoma (Lipton, 2004).

Relevance of excitotoxicity to glaucoma

Whether excitotoxicity participates in the pathophysiology of glaucoma has been a topic of much debate. One contested study detected higher levels of glutamate in the vitreous of glaucoma patients than in controls (Dreyer et al., 1996), while other groups have not replicated this finding (Honkanen et al., 2003). Meanwhile, elevated glutamate levels have been observed in ocular tissues in patients with other retinal diseases in which involvement of glutamate toxicity has been suggested, i.e., in the vitreous of patients with proliferative diabetic retinopathy (Ambati et al., 1997) and in aqueous humor of patients with retinal artery occlusion (Wakabayashi et al., 2006). Importantly, however, one need not have elevated levels of glutamate in order to observe a component of excitotoxicity in pathophysiology of glaucoma. For example, since RGCs that are compromised for almost any reason manifest energy failure, the cells will depolarize as the ionic pumps begin to fail. Therefore, the normal block of NMDA receptor-operated

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channels by Mg2+ decreases (positive charges repel, and, as the intracellular side of the RGC membrane becomes more depolarized or positively charged, it will therefore repel the Mg2+ ion normally lodged in the ion channel pore). Hence, sick, depolarized neurons manifest relief from Mg2+ block, rendering the cells susceptible to damage by normal levels of glutamate (Zeevalk and Nicklas, 1992; Lipton, 2003).

Neurons and glia contain a high concentration of intracellular glutamate (B10 mM) for both metabolism and neurotransmission (Lipton and Rosenberg, 1994). However, glutamate stored within cells is not harmful. Only ‘‘extracellular’’ glutamate can cause excitotoxicity via the recep- tor-mediated mechanisms described here. Therefore, localization of glutamate (intracellular or extracellular) is critical, and thus measuring extracellular glutamate levels is much more meaningful than measuring total glutamate contents in tissues. Glutamate levels in the vitreous have been measured under the premise that the vitreous represents the ‘‘extracellular space’’ of the retina. However, the true extracellular space is the ‘‘intercellular’’ space between retinal cells. Because of technical difficulties in measuring such glutamate concentrations in the human retina, no one really knows if there is an elevation of glutamate or not in human patients with glaucoma. Most importantly, however, Hare et al. (2001, 2004a, b) and WoldeMu¨ssie et al. (2002) have shown that an NMDA receptor antagonist can protect RGCs from both histological and electrophysiological correlates of glaucoma in a well-known monkey model of the disease, as discussed below.

How RGCs die in response to elevated intraocular pressure (IOP) in human glaucoma, and whether excitotoxicity is involved, is still a mystery. Furthermore, particularly in Asia, the high prevalence of normal-tension glaucoma, which manifests glaucomatous optic neuropathy without elevated IOP (Shiose et al., 1991; Klein et al., 1992), makes the etiology even more enigmatic. The epicenter of glaucomatous optic neuropathy is proposed to be the optic nerve head or lamina cribrosa, where soft tissues, including RGC axons and blood vessels circulating the optic nerve head, are likely to be compressed as a result of

deformation of the laminar structure (Quigley, 1995, 1999). Currently, a dominant hypothesis accounting for RGC loss in glaucoma is that obstructed retrograde axonal transport at the lamina cribrosa deprives RGCs from neurotrophic factors, leading them to die, as proposed by Quigley (1995). Even in this scenario, excitotoxicity may participate in the pathophysiology of glaucoma by causing secondary RGC death because of glutamate leaking out of injured cells, thereby triggering oxidative and nitrosative stress (Fig. 2, dotted arrow). This mechanism has been described as a final common pathway contributing to neuronal degeneration in many neurological disorders (Lipton and Rosenberg, 1994).

Some authorities also opine that the optic nerve head is ischemic in glaucoma (Flammer and Orgul, 1998; Osborne et al., 2001). If this is true, excitotoxicity can almost certainly contribute to the pathophysiology of glaucoma because glutamate clearance by glia decreases under ischemic condition (Lipton and Rosenberg, 1994; Szatkowski and Attwell, 1994; Billups and Attwell, 1996; Li et al., 1999). In this regard, retinas of glaucoma patients showed significantly lower immunoreactivity of the excitatory amino acid transporter-1 (EAAT-1), an enzyme responsible for glutamate clearance (Naskar et al., 2000). Together with inappropriate release of glutamate from metabolically compromised cells (Zeevalk and Nicklas, 1992; Lipton and Rosenberg, 1994; Szatkowski and Attwell, 1994), elevation of extracellular glutamate concentration may occur within the glaucomatous retina (Fig. 2). We emphasize here again that if Mg2+ block of NMDA receptors is relieved (as depicted in Fig. 1C), excitotoxicity can come into play in RGCs even without elevated glutamate concentrations (Zeevalk and Nicklas, 1992; Lipton, 2003). During ischemia, disruption of energy metabolism would lead to depolarization of RGCs and relieve Mg2+ block of NMDA receptors (Zeevalk and Nicklas, 1992). In addition to depolarization, there are several factors that can impair the Mg2+ block, among which is mechanical stress. Sublethal stretch caused almost complete loss of the Mg2+ block in cortical neurons (Zhang et al., 1996) and rendered them vulnerable to low concentrations of NMDA (Arundine et al.,

2003), and this affect of stretch in activating NMDA receptors has been replicated on RGCs (R.H. Farkas and S.A. Lipton, unpublished). Other reported factors that impede the Mg2+ block of the NMDA receptors are axonal injury (Furukawa et al., 2000) and inflammation (Guo and Huang, 2001). In conclusion, Mg2+ blockade

of NMDA receptors may be relieved in the face of ischemia, mechanical stress, axonal injury, and inflammation, all of which have been proposed as mechanisms that are associated with high IOP and RGC damage in glaucoma (Quigley, 1995, 1999; Flammer and Orgul, 1998; Osborne et al., 2001; Tezel and Wax, 2004; Burgoyne et al., 2005) (Fig. 2).