Glutamate is one of the 20 amino acids forming part of proteins. It is critical for cell function and is not an essential nutrient, because in man it can be synthesized from other compounds. It is the classic excitatory neurotransmitter in the human cortex. Its role as a neurotransmitter is mediated by the stimulation of specific receptors, called glutamate receptors, which are classified into ionotropic (ion channel) and metabotropic receptors (seven transmembrane G protein coupled domains). All neurons contain glutamate, but only a few use it as a neurotransmitter. Glutamate is potentially excitotoxic (Fig. 12.1). Whereas a variety of neurotransmitters could potentially trigger excitotoxic cell injury, glutamate is thought to be the primary contributor because of its potent effect on increasing intracellular calcium through ionotropic receptors [34]. Therefore, a complex machinery to regulate levels is active. In this regard, and of special interest, the central role played by NO in the CNS has been emphasized in the current literature.

In CNS, NO can be originated from at least four different sources: the endothelium of cerebral vessels, the immunostimulated microglia and astrocytes, the nonadrenergic noncholinergic nerve, and the glutamate neuron [35]. It should be noted that the highest stimulus for the release of NO is the activation of NMDA receptors by glutamate. Also, the release of NO can also be elicited by non-NMDA receptors for glutamate, as well as receptors for acetylcholine, angiotensin, bradykinin, serotonin (5-hydroxytryptamine; 5-HT), neurotensin, and endothelin [36].

An original report by Dawson et al. established that NO mediates the neurotoxicity of glutamate [37]. The authors proposed free radical formation linked to neurotoxicity, and NO is a reactive free radical. According to this, a growing body of evidence suggests involvement of oxidative stress, inflammation, and apoptosis in neurodegenerative diseases [38–41] (Fig. 12.1). Moreover, apoptosis is a regulated process inherent to the normal cellular brain development and/or maintenance; nevertheless a clear deregulation of the mitochondrial respiratory mechanism has been described in patients with neurodegeneration associated to an increase of oxidative stress [42–44].

Toxicity mediated by NO has been controversial. In this sense, Dr. Kiedrowski suggested that the neuroprotective properties of a NO donor such as sodium nitroprusside (SNP) on glutamate- and NMDA-induced neurotoxicity are not due to the release of NO and activation of guanylate cyclase, but are determined by the ferrocyanide portion of the SNP molecule [45]. NO was demonstrated to afford protection from NMDA receptor-mediated neurotoxicity. This pathway for NO regulation of physiological function is not via cGMP, but instead involves reactions with membrane-bound thiol groups on the NMDA receptor-channel complex [46].

NO can react with superoxide to yield peroxynitrate, which is extremely reactive [47]. In models of macrophage-mediated cytotoxicity, NO can complex with the iron–sulfur center of enzymes to inactivate them [48]. Because several of these enzymes are in the mitochondrial electron-transport complex, NO can inhibit mitochondrial respiration, diminishing the ability of the cells to deal with oxidative stress. Specifically, high concentrations of NO irreversibly inhibit complexes I, II, III, IV, and V in the mitochondrial respiratory chain (Fig. 12.1), whereas physiological levels of NO reversibly reduce cytochrome oxidase [49]. Also, further evidence was found in a study on manganese neurotoxicity. Manganese is sequestered in mitochondria, where it inhibits oxidative phosphorylation. The exposure to manganese results in important changes. They include decreased uptake of glutamate. Increased densities of binding sites for the “peripheral-type” benzodiazepine receptor may also be observed. This is a class of receptor localized in the mitochondria of astrocytes, and involved in oxidative metabolism and mitochondrial proliferation. An increased uptake of L-arginine, a precursor of NO, together with increased expression of the inducible form of NOS (iNOS) has also been reported. Accordingly, potential consequences include failure of energy metabolism, production of reactive oxygen species (ROS), and increased extracellular glutamate concentration with excitotoxicity effects [50] (Fig. 12.1).

The mechanisms of neurotoxicity involve activation of NMDA receptors by glutamate, production of NO by nNOS and iNOS, oxidative injury to DNA, and activation of the DNA damage-sensing enzyme poly (ADP-ribose) polymerase (PARP). In this sense, the translocation of a mitochondrial protein apoptosis-inducing factor (AIF) from mitochondria to the nucleus depends on PARP activation, and plays an important role in excitotoxicity-induced cell death [51]. In addition, the accumulation of calcium into mitochondria may play a key role as a trigger to mitochondrial pathology. In the case of calcium overload in neurons, the neurotoxicity of glutamate depends on mitochondrial calcium uptake, but the toxicity to mitochondria also requires the generation of NO. The calcium increase mediated by NMDA receptor activation is thus associated with NO, and the combination leads to the collapse of mitochondrial membrane potential followed by cell death [52].

It is clear that glutamate neurotoxicity is mediated, at least in part, by NO and mitochondrial damage. However, recently a closely related new finding has been postulated. These reports indicate that heat shock protein 70 (Hsp70) upregulation may provide protection in depression by downregulation of iNOS protein expression through suppression of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation [53]. This was validated by Liu et al., who used an in-vitro spinal cord injury model induced by glutamate treatment. Here, treatment with allicin (an organosulfur compound obtained from garlic) significantly attenuated glutamate-induced lactate dehydrogenase (LDH) release, loss of cell viability, and apoptotic neuronal death. Allicin decreased the expression of iNOS following glutamate exposure. Moreover, allicin treatment significantly increased the expression of Hsp70 [54].

Heat shock proteins (HSP) are a shock-induced family of proteins, whose most prominent members are a group of molecules dedicated to maintaining the function of other proteins. Interestingly, after being exposed to heat shock, typical proinflammatory agonists modify the heat shock-induced transcriptional program and expression of HSP genes, suggesting a complex reciprocal regulation between the inflammatory pathway and that of the heat shock response. The specific task of Hsp70, the most widespread and highly conserved HSP, is to protect against inflammation through multiple mechanisms. Hsp70 modulates inflammatory response, as well as downregulating the nuclear factor kappa-light-chain-enhancer of activated B cells. Also, a decreased expression of renal Hsp70 may contribute to the activation of the toll-like receptor 4-initiating inflammatory signal pathway. In addition, several studies have revealed that Hsp70 is involved in the regulation of angiotensin II, a peptide with proinflammatory activity. Increased inflammatory response is generated by nicotinamide adenine dinucleotide phosphate oxidase (NADPH), following activation by angiotensin II. Also, Hsp70 protects the epithelium by modulation of NADPH, a fundamental step in the pro-inflammatory mechanism [55].

Inflammation is present in many diseases, such as diabetes, obesity, metabolic syndrome, impaired glucose tolerance, hypertension, cardiac disease, and CNS disease [19, 56]. Inflammation is connected to mitochondrial dysfunction, overproduction of oxidants, and an over-activation of the renin–angiotensin system linked to NADPH oxidase activity [57]. In addition, NO is also associated with inflammation linked to mitochondrial dysfunction. Moreover, and as mentioned above, reduced NO release induces Hsp70 expression [54], mediating beneficial effects against oxidative stress injury, inflammation, and apoptosis [19, 58]. Curiously, 15 years ago, an elevated expression of the genes encoding Hsp70 linked to apoptosis or necrosis induced by glutamate, was proposed [59]. Later, Hsp70 was suggested as a molecular marker of neurotoxicity [60]. Accordingly, some chaperones such as the members of the Hsp70 family also modulate polyglutamine (polyQ) aggregation and suppress its toxicity. These findings suggested that an imbalance between the neuronal chaperone capacity and the production of potentially dangerous polyQ proteins may trigger the onset of polyQ disease [61]. The formation of insoluble protein aggregates in neurons is a hallmark of neurodegenerative diseases caused by proteins with expanded polyQ repeats. In addition, the more frequent amyloid-related neurodegenerative diseases are caused by a gain of toxic function of misfolded proteins. Toxicity in these disorders may result from an imbalance between normal chaperone capacity and production of dangerous protein species. Increased chaperone expression can suppress the neurotoxicity of these molecules, suggesting possible therapeutic strategies [62]. Moreover, the effects of the Hsp70 were investigated in tau oligomers and tau toxicity linked to neurodegenerative disease. The authors illustrated that Hsp70 preferentially binds to tau oligomers rather than filaments and prevents the anterograde fast axonal transport inhibition observed with a mixture of both forms of aggregated tau [63]. All this evidence strengthens the idea that a reduced NO release linked to induced Hsp70 expression can mediate beneficial effects against oxidative stress injury, inflammation, and apoptosis, during neurodegenerative and neurotoxicity diseases [64] (Fig. 12.1). In addition, abnormalities in NO signaling may constitute a trait-marker related to neuroinflammation, which could be explored for novel therapeutic targets [65].

Finally, the etiology of main neurodegenerative diseases is still unknown, but increasing evidence suggests that glutamate and mitochondria are two key players in the oxidative stress process that underlie these illnesses. Moreover, an emergent role of NO pathways linked to mitochondrial dysfunction has been proposed. It is of particular interest to current understanding. These findings were discussed in the section concerning neurotoxicity from glutamate-induced apoptosis. Taken together, evidence suggests that NO pathways modulation could prevent oxidative damage to neurons by apoptosis inhibition. The discussion remains open on emergent aspects of nitric oxide-mediated signaling in the brain, and how they can be related to neurotoxicity as well as to neurodegenerative diseases development.