Redox Regulation of Protein Function via Cysteine S-Nitrosylation and Its Relevance to Neurodegenerative Diseases

Debilitating neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD), can be attributed to neuronal cell damage in specific brain regions. An important hallmark of these diseases is increased oxidative and nitrosative stress that occurs via overproduction of highly reactive free radicals known as reactive oxygen species (ROS) and reactive nitrogen species (RNS). These molecules are normally removed by cellular antioxidant systems. Under physiological conditions, ROS/RNS are present at low levels, mediating several neurotrophic and neuroprotective signaling pathways. In contrast, under pathological conditions, there is a pronounced increase in ROS/RNS generation, impairing normal neurological function. Nitric oxide (NO) is one such molecule that functions as a signaling agent under physiological conditions but causes nitrosative stress under pathological conditions due to its enhanced production. As first reported by our group and colleagues, the toxic effects of NO can be in part attributed to thiol S-nitrosylation, a posttranslational modification of cysteine residues on specific proteins. Here, we review several reports appearing over the past decade showing that S-nitrosylation of an increasing number of proteins compromises important cellular functions, including mitochondrial dynamics, endoplasmic reticulum (ER) protein folding, and signal transduction, thereby promoting synaptic damage, cell death, and neurodegeneration

S-Nitrosylation of parkin as a novel regulator of p53-mediated neuronal cell death in sporadic Parkinson¿s disease

Abstract Background Mutations in the gene encoding parkin, a neuroprotective protein with dual functions as an E3 ubiquitin ligase and transcriptional repressor of p53, are linked to familial forms of Parkinson’s disease (PD). We hypothesized that oxidative posttranslational modification of parkin by environmental toxins may contribute to sporadic PD. Results We first demonstrated that S-nitrosylation of parkin decreased its activity as a repressor of p53 gene expression, leading to upregulation of p53. Chromatin immunoprecipitation as well as gel-shift assays showed that parkin bound to the p53 promoter, and this binding was inhibited by S-nitrosylation of parkin. Additionally, nitrosative stress induced apoptosis in cells expressing parkin, and this death was, at least in part, dependent upon p53. In primary mesencephalic cultures, pesticide-induced apoptosis was prevented by inhibition of nitric oxide synthase (NOS). In a mouse model of pesticide-induced PD, both S-nitrosylated (SNO-)parkin and p53 protein levels were increased, while administration of a NOS inhibitor mitigated neuronal death in these mice. Moreover, the levels of SNO-parkin and p53 were simultaneously elevated in postmortem human PD brain compared to controls. Conclusions Taken together, our data indicate that S-nitrosylation of parkin, leading to p53-mediated neuronal cell death, contributes to the pathophysiology of sporadic PD

Role of sulfiredoxin as a peroxiredoxin-2 denitrosylase in human iPSC-derived dopaminergic neurons

Recent studies have pointed to protein S-nitrosylation as a critical regulator of cellular redox homeostasis. For example, S-nitrosylation of peroxiredoxin-2 (Prx2), a peroxidase widely expressed in mammalian neurons, inhibits both enzymatic activity and protective function against oxidative stress. Here, using in vitro and in vivo approaches, we identify a role and reaction mechanism of the reductase sulfiredoxin (Srxn1) as an enzyme that denitrosylates (thus removing -SNO) from Prx2 in an ATP-dependent manner. Accordingly, by decreasing S-nitrosylated Prx2 (SNO-Prx2), overexpression of Srxn1 protects dopaminergic neural cells and human-induced pluripotent stem cell (hiPSC)-derived neurons from NO-induced hypersensitivity to oxidative stress. The pathophysiological relevance of this observation is suggested by our finding that SNO-Prx2 is dramatically increased in murine and human Parkinson's disease (PD) brains. Our findings therefore suggest that Srxn1 may represent a therapeutic target for neurodegenerative disorders such as PD that involve nitrosative/oxidative stress

Role of sulfiredoxin as a peroxiredoxin-2 denitrosylase in human iPSC-derived dopaminergic neurons

Recent studies have pointed to protein S-nitrosylation as a critical regulator of cellular redox homeostasis. For example, S-nitrosylation of peroxiredoxin-2 (Prx2), a peroxidase widely expressed in mammalian neurons, inhibits both enzymatic activity and protective function against oxidative stress. Here, using in vitro and in vivo approaches, we identify a role and reaction mechanism of the reductase sulfiredoxin (Srxn1) as an enzyme that denitrosylates (thus removing -SNO) from Prx2 in an ATP-dependent manner. Accordingly, by decreasing S-nitrosylated Prx2 (SNO-Prx2), overexpression of Srxn1 protects dopaminergic neural cells and human-induced pluripotent stem cell (hiPSC)-derived neurons from NO-induced hypersensitivity to oxidative stress. The pathophysiological relevance of this observation is suggested by our finding that SNO-Prx2 is dramatically increased in murine and human Parkinson’s disease (PD) brains. Our findings therefore suggest that Srxn1 may represent a therapeutic target for neurodegenerative disorders such as PD that involve nitrosative/oxidative stress

Transgenic neuronal nitric oxide synthase expression induces axotomy-like changes in adult motoneurons

Dysregulation of protein expression, function and/or aggregation is a hallmark of a number of neuropathological conditions. Among them, upregulation and/or de novo expression of the neuronal isoform of nitric oxide (NO) synthase (nNOS) commonly occurs in diverse neurodegenerative diseases and in axotomized motoneurons. We used adenoviral (AVV) and lentiviral (LVV) vectors to study the effects of de novo nNOS expression on the functional properties and synaptic array of motoneurons. AVV-nNOS injection into the genioglossus muscle retrogradely transduced neonatal hypoglossal motoneurons (HMNs). Ratiometric real-time NO imaging confirmed that transduced HMNs generated NO gradients in brain parenchyma (space constant: ∼12.3 μm) in response to a glutamatergic stimulus. Unilateral AVV-nNOS microinjection in the hypoglossal nucleus of adult rats induced axotomy-like changes in HMNs. Specifically, we found alterations in axonal conduction properties and the recruitment order of motor units and reductions in responsiveness to synaptic drive and in the linear density of synaptophysin-positive puncta opposed to HMN somata. Functional alterations were fully prevented by chronic treatment with nNOS or soluble guanylyl cyclase inhibitors. Synaptic and functional changes were also completely avoided by prior intranuclear injection of a neuron-specific LVV system for miRNA-mediated nNOS knock-down (LVV-miR-shRNA/nNOS). Furthermore, synaptic and several functional changes evoked by XIIth nerve injury were to a large extent prevented by intranuclear administration of LVV-miR-shRNA/nNOS. We suggest that nNOS up-regulation creates a repulsive NO gradient for synaptic boutons underlying most of the functional impairment undergone by injured motoneurons. This further strengthens the case for nNOS targeting as a plausible strategy for treatment of peripheral neuropaties and neurodegenerative disorders

Isogenic Human iPSC Parkinson’s Model Shows Nitrosative Stress-Induced Dysfunction in MEF2-PGC1α Transcription

Parkinson’s disease (PD) is characterized by loss of A9 dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). An association has been reported between PD and exposure to mitochondrial toxins, including environmental pesticides paraquat, maneb, and rotenone. Here, using a robust, patient-derived stem cell model of PD allowing comparison of A53T α-synuclein (α-syn) mutant cells and isogenic mutation-corrected controls, we identify mitochondrial toxin-induced perturbations in A53T α-syn A9 DA neurons (hNs). We report a pathway whereby basal and toxin-induced nitrosative/oxidative stress results in S-nitrosylation of transcription factor MEF2C in A53T hNs compared to corrected controls. This redox reaction inhibits the MEF2C-PGC1α transcriptional network, contributing to mitochondrial dysfunction and apoptotic cell death. Our data provide mechanistic insight into gene-environmental interaction (GxE) in the pathogenesis of PD. Furthermore, using small-molecule high-throughput screening, we identify the MEF2C-PGC1α pathway as a therapeutic target to combat PD.Parkinson Society Canada (Fellowship)United Mitochondrial Disease Foundation (grant)National Institutes of Health (U.S.) (NIH grant P01 HD29587)National Institutes of Health (U.S.) (NIH grant P01 ES016738)National Institutes of Health (U.S.) (NIH grant P30 NS076411)National Institutes of Health (U.S.) (NIH grant R37 CA084198