Application of novel therapeutic agents for CNS injury: NAAG peptidase inhibitors

Glutamate excitotoxicity is a significant determinant of traumatic brain injury (TBI) pathophysiology. Elevated glutamate can damage neurons through activation of various glutamate receptors. Excessive extracellular glutamate also initiates astrocyte pathology through excessive uptake of Na+ through sodium-glutamate co-transporters. This chapter discusses a novel strategy for reducing glutamate toxicity following TBI by inhibiting the cleavage of N-acetylaspartylglutamate (NAAG) into N-acetylaspartate (NAA) and glutamate. NAAG is an abundant peptide found in the brain and is released from neurons after intense depolarization and functions as a selective agonist at metabotropic glutamate receptor subtype 3 (mGluR3), which is located on both neurons and astrocytes. NAAG is catabolized into NAA and glutamate by a specific carboxypeptidases. NAAG could play a significant role in modulating glutamate excitotoxicity if its rapid catabolism can be inhibited thereby conferring protection to the traumatized brain in several ways. First, NAAG reduces excessive glutamate release by activation of presynaptic mGluR3 autoreceptors. Second, by inhibiting the cleavage of NAAG into NAA and glutamate a secondary source of synaptic glutamate could be diminished. Third, activation of mGluR3 on astrocytes increases the expression of glutamate transporters thereby facilitating removal of excess glutamate from the synapse. Fourth, inhibiting the accumulation of the NAAG cleavage product, NAA, reduces NAA-Na+ co-transport and subsequent astrocyte Na+ overload. Overload of [Na+]i can initiate astrocyte pathology that subsequently impacts negatively on surrounding neurons. Thus, inhibition of carboxypeptidases represents a novel strategy for reducing glutamate excitotoxicity following TBI through multiple mechanisms. NAAG peptidase inhibition could provide new and important insights into glutamate excitotoxicity and lead to important insights into the dynamics of neuron-astrocyte interactions in TBI pathophysiology.An estimated 1.7 million persons per year sustain a TBI in the United States resulting in more than 230,000 hospitalizations and 50,000 deaths (Faul et al., 2010). Survivors of TBI are often left with long-term disability (approximately 85,000 new cases per year) encompassing a broad spectrum of physical, neurological, and psychological dysfunctions such as memory impairment and difficulties with attention and concentration (Thurman and Guerrero, 1999; Thurman et al., 1999). The annual economic cost to society for the care of head-injured patients has been estimated to exceed $25 billion (Goldstein, 1990). TBI research to date, including glutamate excitotoxicity pathways, has focused almost exclusively on the pathophysiology of injured neurons with very little attention paid to glial cells or the interaction between glia and neurons. Astrocytes, which play an important role in normal brain function, increasingly appear to play a critical role in perturbed brain functions associated with seizures and ischemia brain injury (Vernadakis 1996; Vesce et al., 1999). This chapter examines a novel method of reducing the damaging effects of elevated extracellular glutamate by manipulating a putative endogenous peptide neurotransmitter, N-acetylaspartylglutamate (NAAG). The role of NAAG in exogenous and endogenous brain cellular protection involves the interdependent nature of neuronal glial interactions and the critical involvement of glutamate excitotoxicity initiated by TBI.Previous therapeutic strategies targeting excitotoxicity in central nervous system (CNS) injury have focused on pharmacological blockade of various ionotropic and metabotropic glutamate receptors. Although laboratory results have often been impressive, translation into clinical applications have been overwhelmingly disappointing (Bullock et al., 1999; Narayan et al., 2002). However, a general perception from laboratory experiments is that excessive glutamate is indeed damaging to neurons (Bullock et al., 1999). This chapter discusses a novel strategy to reduce glutamate toxicity by simultaneously enhancing levels of an endogenous neurotransmitter peptide, NAAG, while reducing its cleavage products, N-acetylaspartate (NAA) and glutamate, through application of NAAG peptidase inhibitors. This approach is designed to reduce excitotoxicity through multiple mechanisms acting in concert.Three pathways of the NAAG neuropeptide story are depicted in Figure 38.1. The first pathway (highlighted in green) depicts how NAAG reduces glutamate release by activation of presynaptic metabotropic glutamate receptor subtype 3 (mGluR3) autoreceptors. This is based on data demonstrating that inhibition of NAAG cleavage elevates levels of extracellular NAAG and reduces extracellular levels of glutamate after TBI (Zhong et al., 2005). Because NAAG is hydrolyzed into NAA and glutamate, the first pathway also depicts how NAAG cleavage provides a secondary source of extracellular glutamate.A second pathway (highlighted in red in the figure) depicts elevated concentrations of NAAG peptide increasing glutamate uptake in astrocytes after TBI as a result of mGluR3 receptor activation on astrocytes. There is an established relationship between NAAG activation of mGluR3 on astrocytes and increased glutamate transport (Aronica et al., 2003; Yao et al., 2005). The expression and kinetics of glutamate transporters could be potentially increased by NAAG activating mGluR3 on astrocytes (Aronica et al., 2003). Thus, the extent of cellular protection afforded by NAAG peptidase inhibitors may not simply be because of reduced glutamate release, but also because of NAAG-induced enhancement of glutamate transport into astrocytes.A third pathway (highlighted in blue in the figure) depicts the role of NAA (a product of NAAG cleavage by NAAG peptidase) in astrocyte pathology after TBI. NAAG is exclusively stored and released in neurons, whereas NAAG peptidase is exclusively found on the surface of astrocytes (Neale et al., 2000). NAA is preferentially and overwhelmingly taken up by sodium-dependent transporters located on astrocytes (Sager et al., 1999b). The excessive NAA/Na+ cotransport into astrocytes after TBI initiates a pathological cascade in astrocytes related to elevated [Na+]i. Glutamate/Na+ cotransporters (GLT-1 and GLAST) located on astrocytes also initiates a Na+-dependent pathological cascade in astrocytes that eventually impacts upon survival of nearby neurons (Floyd et al., 2005).NAAG likely functions as an endogenous protective peptide through multiple pathways involving both neurons and astrocytes. However, NAAG is short-lived in the synapse because of its rapid catabolism by NAAG peptidases (glutamate carboxypeptidase II [GCP-II] and GCP-III), which diminishes the beneficial effects of NAAG. Application of NAAG peptidase inhibitors represents a novel method of enhancing the endogenous protective effects of NAAG by enhancing and sustaining concentrations of NAAG in the synapse.