Targeting excitotoxic/free radical signaling pathways for therapeutic intervention in glaucoma

Introduction

Glutamate is the predominant excitatory neurotransmitter in the central nervous system. However, the presence of glutamate at excessive concentration or for excessive periods of time can excite neurons to death. This phenomenon was first discovered in the retina (Lucas and Newhouse,

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1957) and later named ‘‘excitotoxicity’’ (Olney and Ho, 1970). Excitotoxicity has been thought to participate in etiology of various neurological disorders, ranging from acute insults (e.g., stroke, hypoglycemia, trauma, and epilepsy) to chronic neurodegenerative diseases (e.g., Huntington’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis, and human immunodeficiency virus [HIV]-associated dementia): glaucoma may possibly be among them (Choi, 1988; Lipton, 1993, 2001, 2003, 2004; Lipton and Rosenberg, 1994; Dreyer and Lipton, 1999). In this chapter, we will

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describe mechanistic insights of excitotoxicity and how excitotoxicity can fit into pathogenesis of glaucoma, at least in part. Thereafter, possible therapeutic interventions to treat glaucoma by interrupting excitotoxic cascades will be discussed.

Channel properties of NMDA receptors correlated with excitotoxicity

The excitatory amino acid, glutamate (glutamic acid), elicits neuronal signaling by binding to glutamate receptors. The glutamate receptors are divided into two major categories, the ionotropic (conducting ions) and metabotropic (triggering biochemical signaling). Excitotoxicity is mediated predominantly through the ionotropic receptors, which comprise of three classes of receptors (a-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid [AMPA] receptors, kainate receptors, and N- methyl-D-aspartate [NMDA] receptors). All these are ligand-gated ion channels that normally allow cations to enter into a cell upon ligand binding. Mammalian retinal ganglion cells (RGCs) express all three ionotropic receptors (Aizenman et al., 1988), and expression of NMDA receptors in the retina is found on RGCs and subsets of amacrine

cells (Brandstatter et al., 1994; Hartveit et al., 1994; Grunder et al., 2000). Although AMPA and kainate receptors can also contribute to excitotoxicity, NMDA receptors appear to play a prominent role (Hahn et al., 1988; Sucher et al., 1991).

NMDA receptors are probably a tetramer of subunits, consisting of an obligatory NR1 subunit plus NR2A-D subunits and possibly modulatory NR3A-B subunits. NR1 and NR3 subunits have glycine-binding sites. NR2 subunits have glutamate-binding sites. Although the NR1 subunit is mandatory to form functional NMDA receptors, agonist binding to the NR2 subunits is mandatory for functional activity of NMDA receptors responding to glutamate. Upon binding of glycine and glutamate (or NMDA under experimental conditions), a neuron becomes depolarized and consequently allows influx of Ca2+ and Na+ into the cell through the NMDA receptor-operated channels. Among the three classes of ionotropic glutamate receptors, the NMDA receptors are the most permeable to Ca2+, which, if fluxed excessively, signals downstream events leading to cell death (Lipton and Rosenberg, 1994).

NMDA receptors have important modulatory sites that can regulate the ion channel activity

of the receptors. Among these are the Mg2+sites within the channel pore and the S-nitrosylation (redox) sites (Sullivan et al., 1994; Lipton et al., 1998, 1999; Choi et al., 2000). We have used these targets for therapeutic intervention to block excitotoxicity, as discussed later in this

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chapter. Mg2+ serves as an endogenous negative regulator of NMDA receptors. At resting membrane potentials of healthy neurons (approximately 70 mV), Mg2+ blocks NMDA receptor-operated channels (Fig. 1A). This Mg2+ block is controlled in a voltage-dependent manner

Na+ influx through the now unblocked NMDA receptor-coupled channels (Fig. 1B). Under physiological conditions, the Mg2+ block soon recovers during repolarization, thus only allowing

is compromised by ischemia or injury, neurons become depolarized spontaneously, thus removing Mg2+ block and rendering NMDA receptors abnormally active, particularly extrasynaptic NMDA receptors because of the location of extracellular glutamate under these pathological conditions (Zeevalk and Nicklas, 1992; Hardingham and Bading, 2003; Lipton, 2006) (Fig. 1C).

Excitotoxicity is a result of overactivation of the glutamate receptors, which can be initiated by the elevated extracellular glutamate concentration or hyperactivity of receptors in the presence of normal glutamate levels. Elevation of extracellular glutamate concentration will occur, for example, after ischemia in the central nervous system, which causes enormous disruption of energy metabolism in both neurons and glia. Under these circumstances, glutamate is not cleared properly by glutamate transporters, which normally take it up mainly into glia, and even reversal of glutamate transport can occur, resulting in increased release (Zeevalk and Nicklas, 1992; Lipton and Rosenberg, 1994; Szatkowski and Attwell, 1994; Billups and Attwell, 1996; Li et al., 1999). Hyperactivity of NMDA receptors in the face of normal glutamate levels can also occur in ischemic nervous tissue because neurons lose their ionic homeostasis and become depolarized. Depolarization relieves Mg2+ block of NMDA receptors (Mayer et al., 1984; Nowak et al., 1984; Zeevalk and Nicklas, 1992). Thus, in the absence of physiological Mg2+ block, NMDA receptorcoupled channels become abnormally active even in the absence of elevated extracellular glutamate concentration.

Downstream signaling cascades after overactivation of NMDA receptors

Overactivation of NMDA receptors triggers an excessive Ca2+ influx into neurons, initiating cell death pathways (Lipton and Rosenberg, 1994) (Fig. 1D). As a consequence of the increase in intracellular Ca2+ concentration ([Ca2+]i) and subsequent Ca2+ entry into mitochondria, the mitochondrial membrane potential depolarizes (Ankarcrona et al., 1995; Green and Reed, 1998). Depolarized mitochondria release various bioactive substances into the cytosol. Cytochrome c released from mitochondria activates caspases, which play an important role in apoptosis through DNA fragmentation (Green and Reed, 1998). Apopto- sis-inducing factor (AIF) is another factor released from mitochondria and also contributes to DNA damage (Yu et al., 2003). Mitochondria are also major sources of reactive oxygen species (ROS), and NMDA stimulation, in fact, causes ROS production in cultured cerebrocortical neurons (Lafon-Cazal et al., 1993; Tenneti et al., 1998).

Calmodulin (CaM) activated by elevation of [Ca2+]i triggers synthesis of nitric oxide (NO) via neuronal NO synthase (nNOS) (Dawson et al., 1991, 1993; Lipton and Rosenberg, 1994; Lipton, 2006), which is physically tethered to NMDA receptors via interaction with postsynaptic density95 (PSD-95) protein linked to NR2 subunits, predominantly the NR2B subunit (Kornau et al., 1995; Christopherson et al., 1999; Sattler et al., 1999). NO is involved in many chemical reactions with a great variety of molecules. For example, S-nitrosylation (a chemical reaction representing transfer of NO to the thiol or sulfhydryl group of a critical cysteine residue) regulates the biological activity of many proteins (Lipton et al., 1993; Hess et al., 2005). Depending on the protein, S-nitrosylation may either stimulate or inhibit activity and lead to either neuronal death or survival (Lipton, 1999; Nakamura and Lipton, 2007). Thus, NO can be both neurodestructive and neuroprotective. NO also reacts with ROS to form highly toxic peroxynitrite (ONOO ), which can injure cells by DNA damage, lipid peroxidation, protein oxidation/nitration, and other mechanisms