At physiological levels, nitric oxide (NO) contributes to the maintenance of normal neuronal activity and survival, thus serving as an important regulatory mechanism in the central nervous system. By contrast, accumulating evidence suggests that exposure to environmental toxins or the normal aging process can trigger excessive production of reactive oxygen/nitrogen species (such as NO), contributing to the etiology of several neurodegenerative diseases. We highlight here protein S-nitrosylation, resulting from covalent attachment of an NO group to a cysteine thiol of the target protein, as a ubiquitous effector of NO signaling in both health and disease. We review our current understanding of this redox-dependent post-translational modification under neurodegenerative conditions, and evaluate how targeting dysregulated protein S-nitrosylation can lead to novel therapeutics.

Normal mitochondrial dynamics consist of fission and fusion events giving rise to new mitochondria, a process termed mitochondrial biogenesis. However, several neurodegenerative disorders manifest aberrant mitochondrial dynamics, resulting in morphological abnormalities often associated with deficits in mitochondrial mobility and cell bioenergetics. Rarely, dysfunctional mitochondrial occur in a familial pattern due to genetic mutations, but much more commonly patients manifest sporadic forms of mitochondrial disability presumably related to a complex set of interactions of multiple genes (or their products) with environmental factors (G × E). Recent studies have shown that generation of excessive nitric oxide (NO), in part due to generation of oligomers of amyloid-β (Aβ) protein or overactivity of the NMDA-subtype of glutamate receptor, can augment mitochondrial fission, leading to frank fragmentation of the mitochondria. S-Nitrosylation, a covalent redox reaction of NO with specific protein thiol groups, represents one mechanism contributing to NO-induced mitochondrial fragmentation, bioenergetic failure, synaptic damage, and eventually neuronal apoptosis. Here, we summarize our evidence in Alzheimer’s disease (AD) patients and animal models showing that NO contributes to mitochondrial fragmentation via S-nitrosylation of dynamin-related protein 1 (Drp1), a protein involved in mitochondrial fission. These findings may provide a new target for drug development in AD. Additionally, we review emerging evidence that redox reactions triggered by excessive levels of NO can contribute to protein misfolding, the hallmark of a number of neurodegenerative disorders, including AD and Parkinson’s disease. For example, S-nitrosylation of parkin disrupts its E3 ubiquitin ligase activity, and thereby affects Lewy body formation and neuronal cell death.

Several chronic neurodegenerative disorders manifest deposits of misfolded or aggregated proteins. Genetic mutations are the root cause for protein misfolding in rare families, but the majority of patients have sporadic forms possibly related to environmental factors. In some cases, the ubiquitin-proteasome system or molecular chaperones can prevent accumulation of aberrantly folded proteins. Recent studies suggest that generation of excessive nitric oxide (NO) and reactive oxygen species (ROS), in part due to overactivity of the NMDA-subtype of glutamate receptor, can mediate protein misfolding in the absence of genetic predisposition. S-Nitrosylation, or covalent reaction of NO with specific protein thiol groups, represents one mechanism contributing to NO-induced protein misfolding and neurotoxicity. Here, we present evidence suggesting that NO contributes to protein misfolding via S-nitrosylating protein-disulfide isomerase or the E3 ubiquitin ligase parkin. We discuss how memantine/NitroMemantine can inhibit excessive NMDA receptor activity to ameliorate NO production, protein misfolding, and neurodegeneration.

The prevalence of neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD), is currently a major public health concern due to the lack of efficient disease-modifying therapeutic options. Recent evidence suggests that mitochondrial dysfunction and nitrosative/oxidative stress are key common mediators of pathogenesis. In this review, we highlight molecular mechanisms linking NO-dependent post-translational modifications, such as cysteine S-nitrosylation and tyrosine nitration, to abnormal mitochondrial metabolism. We further discuss the hypothesis that pathological levels of NO compromise brain energy metabolism via aberrant S-nitrosylation of key enzymes in the tricarboxylic acid (TCA) cycle and oxidative phosphorylation, contributing to neurodegenerative conditions. A better understanding of these pathophysiological events may provide a potential pathway for designing novel therapeutics to ameliorate neurodegenerative disorders.

Significance: Most brains affected by neurodegenerative diseases manifest mitochondrial dysfunction as well as elevated production of reactive oxygen species and reactive nitrogen species (RNS), contributing to synapse loss and neuronal injury. Recent Advances: Excessive production of RNS triggers nitric oxide (NO)-mediated post-translational modifications of proteins, such as S-nitrosylation of cysteine residues and nitration of tyrosine residues. Proteins thus affected impair mitochondrial metabolism, mitochondrial dynamics, and mitophagy in the nervous system. Critical Issues: Identification and better characterization of underlying molecular mechanisms for NO-mediated mitochondrial dysfunction will provide important insights into the pathogenesis of neurodegenerative disorders. In this review, we highlight recent discoveries concerning S-nitrosylation of the tricarboxylic acid cycle enzymes, mitochondrial fission GTPase dynamin-related protein 1, and mitophagy-related proteins Parkin and phosphatase and tensin homolog-induced putative kinase protein 1. We delineate signaling cascades affected by pathologically S-nitrosylated proteins that diminish mitochondrial function in neurodegenerative diseases. Future Directions: Further elucidation of the pathological events resulting from aberrant S-nitrosothiol or nitrotyrosine formation may lead to new therapeutic approaches to ameliorate neurodegenerative disorders.

Autophagy (in the form of macroautophagy) is the major intracellular protein quality control system for removal of damaged organelles and abnormally aggregated proteins. We and others have shown that dysregulated autophagic pathways contribute to accumulation and spread of misfolded proteins in many neurodegenerative disorders, including Parkinson disease (PD) and Lewy body dementia (LBD). Additionally, generation of excessive reactive oxygen and nitrogen species, such as nitric oxide (NO), accelerates neuronal and synaptic damage mediated, at least in part, via aberrant protein S-nitrosylation. Using cell-based models, including human induced pluripotent stem cell (hiPSC)-derived neurons, CRISPR-Cas9 technology, and transgenic PD/LBD mice, plus vetting in human postmortem brains, we found that S-nitrosylation of the autophagic receptor protein SQSTM1/p62 (forming SNO-SQSTM1/p62) inhibits autophagic flux, thus contributing to accumulation of misfolded SNCA/α-synuclein. Consequently, this impairment in autophagy increases extracellular vesicle-dependent secretion and spread of aggregated SNCA. Taken together, our evidence suggests that aberrant formation of SNO-SQSTM1/p62 represents a pathogenic event contributing not only to inhibition of autophagic flux and potentiation of neuronal damage, but also to propagation of α-synucleinopathy between cells in the diseased brain.

Alzheimer's disease (AD) is arguably the most common cause of dementia in the elderly and is marked by progressive synaptic degeneration, which in turn leads to cognitive decline. Studies in patients and in various AD models have shown that one of the early signatures of AD is neuronal hyperactivity. This excessive electrical activity contributes to dysregulated neural network function and synaptic damage. Mechanistically, evidence suggests that hyperexcitability accelerates production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) that contribute to neural network impairment and synapse loss. This review focuses on the pathways and molecular changes that cause hyperexcitability and how RNS-dependent posttranslational modifications, represented predominantly by protein S-nitrosylation, mediate, at least in part, the deleterious effects of hyperexcitability on single neurons and the neural network, resulting in synaptic loss in AD.

Neurodegenerative disorders like Alzheimer's disease and Parkinson's disease are characterized by progressive degeneration of synapses and neurons. Accumulation of misfolded/aggregated proteins represents a pathological hallmark of most neurodegenerative diseases, potentially contributing to synapse loss and neuronal damage. Emerging evidence suggests that misfolded proteins accumulate in the diseased brain at least in part as a consequence of excessively generated reactive oxygen species (ROS) and reactive nitrogen species (RNS). Mechanistically, not only disease-linked genetic mutations but also known risk factors for neurodegenerative diseases, such as aging and exposure to environmental toxins, can accelerate production of ROS/RNS, which contribute to protein misfolding - in many cases mimicking the effect of rare genetic mutations known to be linked to the disease. This review will focus on the role of RNS-dependent post-translational modifications, such as S-nitrosylation and tyrosine nitration, in protein misfolding and aggregation. Specifically, we will discuss molecular mechanisms whereby RNS disrupt the activity of the cellular protein quality control machinery, including molecular chaperones, autophagy/lysosomal pathways, and the ubiquitin-proteasome system (UPS). Because chronic accumulation of misfolded proteins can trigger mitochondrial dysfunction, synaptic damage, and neuronal demise, further characterization of RNS-mediated protein misfolding may establish these molecular events as therapeutic targets for intervention in neurodegenerative diseases.

S-Nitrosylation is well established as an important post-translational regulator in protein function and signaling. However, relatively little is known about its structural and dynamical consequences. We have investigated the effects of S-nitrosylation on the rhodanese domain of the Escherichia coli integral membrane protein YgaP by NMR, X-ray crystallography, and mass spectrometry. The results show that the active cysteine in the rhodanese domain of YgaP is subjected to two competing modifications: S-nitrosylation and S-sulfhydration, which are naturally occurring in vivo. It has been observed that in addition to inhibition of the sulfur transfer activity, S-nitrosylation of the active site residue Cys63 causes an increase in slow motion and a displacement of helix 5 due to a weakening of the interaction between the active site and the helix dipole. These findings provide an example of how nitrosative stress can exert action at the atomic level.

Significance: Physiological concentrations of nitric oxide (NO^•) and related reactive nitrogen species (RNS) mediate multiple signaling pathways in the nervous system. During inflammaging (chronic low-grade inflammation associated with aging) and in neurodegenerative diseases, excessive RNS contribute to synaptic and neuronal loss. "NO signaling" in both health and disease is largely mediated through protein S-nitrosylation (SNO), a redox-based posttranslational modification with "NO" (possibly in the form of nitrosonium cation [NO⁺]) reacting with cysteine thiol (or, more properly, thiolate anion [R-S^-]). Recent Advances: Emerging evidence suggests that S-nitrosylation occurs predominantly via transnitros(yl)ation. Mechanistically, the reaction involves thiolate anion, as a nucleophile, performing a reversible nucleophilic attack on a nitroso nitrogen to form an SNO-protein adduct. Prior studies identified transnitrosylation reactions between glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-nuclear proteins, thioredoxin-caspase-3, and X-linked inhibitor of apoptosis (XIAP)-caspase-3. Recently, we discovered that enzymes previously thought to act in completely disparate biochemical pathways can transnitrosylate one another during inflammaging in an unexpected manner to mediate neurodegeneration. Accordingly, we reported a concerted tricomponent transnitrosylation network from Uch-L1-to-Cdk5-to-Drp1 that mediates synaptic damage in Alzheimer's disease. Critical Issues: Transnitrosylation represents a critical chemical mechanism for transduction of redox-mediated events to distinct subsets of proteins. Although thousands of thiol-containing proteins undergo S-nitrosylation, how transnitrosylation regulates a myriad of neuronal attributes is just now being uncovered. In this review, we highlight recent progress in the study of the chemical biology of transnitrosylation between proteins as a mechanism of disease. Future Directions: We discuss future areas of study of protein transnitrosylation that link our understanding of aging, inflammation, and neurodegenerative diseases. Antioxid. Redox Signal. 35, 531-550.