2 International Journal of Cell Biology Antioxidant mechanisms • Cysteine-based redox regulation Keap1/Nrf2 Hsp90/HSF1 Low ROS/RNS ROS/RNS production ROS signaling NO signaling Neuroprotection High ROS/RNS Oxidative/nitrosative stress Macromolecular damage Protein carbonylation, nitration, and S-nitrosylation Lipid peroxidation DNA double strand break Cellular damage/death Neurodegeneration Figure 1: Imbalance in oxidant production and antioxidant mechanisms contributes to neurodegeneration. Under physiological conditions, antioxidant mechanisms such as cysteine-based redox regulation (Prx, Grx, Trx, glutathione (GSH), etc.), as well as transcriptional pathways represented by Keap1/Nrf2 and Hsp90/HSF1, maintain low concentrations of ROS/RNS in the neurons. These low levels of oxidants activate speciﬁc signaling pathways that subserve normal cell signaling and in fact may be neuroprotective in nature. On the other hand, under pathological situations, including AD and PD, there is a decrease in antioxidant mechanisms and increased oxidant production, eﬀectively creating high levels of ROS/RNS. Oxidative/nitrosative stress generated in this manner can contribute to cell damage and results in neurodegeneration. to this nature, ROS and RNS can react somewhat indiscrim- inately with all classes of biological macromolecules (e.g., protein, lipid, DNA) and cause cellular damage (Figure 1). In this paper, we will speciﬁcally address the eﬀect of nitrosative stress triggered by NO species that react to form protein S- nitrosothiols. It should be noted, however, that NO signaling can result in other types of posttranslational modiﬁcations, such as protein tyrosine nitration and S-glutathionylation, as well as reaction with heme, for example, to activate soluble guanylate cyclase to form cGMP [12]. 2. Nitric Oxide Production and Signaling Cellular production of NO from l-arginine is catalyzed by a family of enzymes known as NO synthases (NOSs). The NOS family consists of endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS) [13], and all three NOS subtypes are expressed in the mammalian brain. For instance, Ca2+-dependent nNOS catalyzes production of NO predominantly in neurons, whereas Ca2+-independent iNOS is primarily (but not exclusively) involved in NO production within microglia and astrocytes [14]. Many excitatory synapses contain N-methyl-d-aspartate- type glutamate receptor- (NMDAR-) operated channels. Activation of these channels results in Ca2+ inﬂux, triggering NO production by nNOS [15]. NO can undergo a number of reactions under normal physiological conditions. For example, NO reacts with soluble guanylate cyclase to produce cyclic GMP (cGMP) [16]. The second messenger cGMP then activates cyclic guanylate kinases (cGKs) [17, 18]. Once activated, cGKs can phosphorylate various physiological substrates in neurons, thereby controlling various important processes, including synaptic transmission and synaptic plasticity [14, 18] (Figure 2). An even more prominent physiological reaction of NO involves the posttranslational modiﬁcation of S-nitrosylation or transfer of an NO group to a critical cysteine sulfhydryl to regulate protein function [19], in some sense analogous to phosphorylation of tyrosine, threonine, or serine residues. However, under pathological conditions, hyperactivation of NMDARs (often located at extrasynaptic or perisynaptic sites) causes massive Ca2+ inﬂux and overproduction of NO [19–21]. Nitrosative stress due to NO overproduction compromises cellular signaling via aberrant protein S-nitrosylation and tyrosine nitration, which can contribute to neuronal cell injury or death [12] (Figure 2). In contrast, it should be noted that several of the proteins that are S-nitrosylated under physiological conditions, for example, the NMDAR itself and GOSPEL foster cell survival [22, 23]. 3. Protein S-Nitrosylation As alluded to above, protein S-nitrosylation is a reversible posttranslational modiﬁcation whereby an NO group is