Ratio of S-nitrosohomocyst(e)ine to homocyst(e)ine or other thiols determines neurotoxicity in rat cerebrocortical cultures.

The physiological activity of many proteins can be regulated by S-nitrosylation or reaction of nitric oxide (NO)-related species with cysteine residues to produce S-nitrosoproteins (S-nitrosothiols). However, S-nitrosothiols, such as S-nitrosocysteine (SNOC) and S-nitrosohomocysteine (SNHC), can also be neurotoxic by generating NO which reacts with endogenous O2- to form peroxynitrite. Additionally, thiols such as cysteine and homocysteine can be neurotoxic by acting as N-methyl-D-aspartate (NMDA) agonists. Paradoxically, we show here that millimolar thiol can protect from acute exposure to micromolar SNHC that is normally neurotoxic. This finding can be best explained by the fact that although S-nitrosothiols undergo homolytic cleavage to produce NO and subsequent neurotoxicity, adding thiol stabilizes S-nitrosothiols, effectively preventing this cleavage. Thus, the equilibrium between thiol and nitrosothiol determines outcome in studies of neuronal degeneration.

Role of caspases in N-methyl-D-aspartate-induced apoptosis in cerebrocortical neurons.

Overactivation of glutamate receptors mediates neuronal death in several acute and chronic neurodegenerative diseases. The intracellular processes underlying this form of death, however, remain poorly understood. Depending on the severity of insult, N-methyl-D-aspartate (NMDA) receptor activation induces either apoptosis or necrosis. Cysteine proteases related to interleukin-1beta-converting enzyme (ICE), recently termed caspases, appear necessary for neuronal apoptosis in vivo and in vitro. To determine whether caspases play a role in NMDA-induced apoptosis, we used two functionally distinct approaches to decrease substrate cleavage by caspases. One is a novel peptide (V-ICEinh) that contains the caspase catalytic site and acts as a pseudoenzyme that binds caspase substrates and prevents their cleavage. The other is a pseudosubstrate peptide (Z-VAD x fmk) that inhibits caspase activity. Pretreatment with either V-ICEinh or Z-VAD-fmk protects cerebrocortical neurons from NMDA-induced apoptosis, suggesting a role for caspases in NMDA-induced apoptosis. To explore the signaling pathways involved, we looked at the effects of NMDA receptor activation on Ca2+ influx, production of reactive oxygen species (ROS), mitochondrial membrane potential, and lipid peroxidation. Neither NMDA-induced Ca2+ influx nor the initial collapse of mitochondrial membrane potential could be prevented by pretreatment with V-ICEinh or Z-VAD x fmk. In contrast, ROS formation and lipid peroxidation were completely blocked by both V-ICEinh and Z-VAD x fmk. Taken together, our results suggest that Ca2+ influx and mitochondrial depolarization occur upstream from caspase activation, whereas ROS formation and lipid peroxidation may be downstream events in the cascade leading to cortical neuronal apoptosis.

Suppression of neuronal apoptosis by S-nitrosylation of caspases.

S-Nitrosylation (reaction of nitric oxide (NO) species with a critical cysteine sulfhydryl) can regulate the physiological activity of proteins, including enzymes, ion channels, G-proteins, and transcription factors. Caspases are a family of interleukin-1beta-converting enzyme-like proteases involved in the signaling pathway to apoptotic cell death, and each member of this enzyme family contains a critical cysteine residue in its active site. Here we show that S-nitrosylation of caspases in human embryonic kidney (HEK)-293 cells and primary cerebrocortical neurons decreases enzyme activity and is associated with protection from apoptosis.

Neurotoxicity associated with dual actions of homocysteine at the N-methyl-D-aspartate receptor.

Severely elevated levels of total homocysteine (approximately millimolar) in the blood typify the childhood disease homocystinuria, whereas modest levels (tens of micromolar) are commonly found in adults who are at increased risk for vascular disease and stroke. Activation of the coagulation system and adverse effects of homocysteine on the endothelium and vessel wall are believed to underlie disease pathogenesis. Here we show that homocysteine acts as an agonist at the glutamate binding site of the N-methyl-D-aspartate receptor, but also as a partial antagonist of the glycine coagonist site. With physiological levels of glycine, neurotoxic concentrations of homocysteine are on the order of millimolar. However, under pathological conditions in which glycine levels in the nervous system are elevated, such as stroke and head trauma, homocysteine's neurotoxic (agonist) attributes at 10-100 microM levels outweigh its neuroprotective (antagonist) activity. Under these conditions neuronal damage derives from excessive Ca2+ influx and reactive oxygen generation. Accordingly, homocysteine neurotoxicity through overstimulation of N-methyl-D-aspartate receptors may contribute to the pathogenesis of both homocystinuria and modest hyperhomocysteinemia.