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ROS production and protein carbonylation in GSNOR-downregulating systems. (a) Cytofluorimetric analyses of H2O2 in siScr and siGSNOR SH-SY5Y cells upon 2′,7′-dihydrodichlorofluorescein (DCF) staining. (b) Cytofluorimetric analyses of H2O2 and O2•- in GSNOR-KO and WT PCN upon 2′,7′-dihydrodichlorofluorescein (DCF) or (c) dihydroethidine (DHE) staining. (d) Western blot analyses of protein carbonyls from mesencephalon obtained from GSNOR-KO and WT brains. Western blots shown are representative of at least n=3 independent experiments that gave similar results. Actin was selected as loading control. Graphs shown represent the mean of data ± SD of n=3 independent experiments, P∗<0.05 and P∗∗<0.01.
Source publication
Oxidative and nitrosative stresses have been reported as detrimental phenomena concurring to the onset of several neurodegenerative diseases. Here we reported that the ectopic modulation of the denitrosylating enzyme
S
-nitrosoglutathione reductase (GSNOR) differently impinges on the phenotype of two SH-SY5Y-based
in vitro
models of neurodegenerati...
Citations
... For instance, we found that GSNOR facilitated antiviral innate immunity by restricting S-nitrosation of TANK-binding kinase 1 [23], and during aging increased GSNOR expression impairs cognitive function and decreases S-nitrosation of CAMK2A (calcium/calmodulin-dependent protein kinase II alpha) [24]. Also, knockdown of GSNOR protected SH-SY5Y cells from 1-Methyl-4-phenylpyridinium (MPP + , a metabolite of MPTP)-induced toxicity [25]. ...
... Briefly, cell lysates of mouse brain tissues, and rat C6 and PC12 cells were prepared, and protein concentration was determined by BCA protein assay kit (Beyotime Institute of Biotechnology, P0012). We used around 25 μg of protein per sample for each assay. Next the protein was separated using 12% or 15% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane (BioRad, L1620177 Rev D), the membrane was soaked with 5% (w/v) skim milk for 2 h at room temperature, then was incubated with primary antibody (Table S1) overnight at 4 • C. The membrane was washed 3 times with TBST (Tris-buffered saline [Servicebio, G0001] with 0.1% Tween 20 [Sigma, P1379]), each time 5 min, followed by incubation with the secondary antibody. ...
... Previous studies reported an aberrant increase of GSNOR protein and enzymatic activity in a variety of diseases including asthma [52], cognitive impairment [24], stroke [46], myocardial infarction [53], and cerebral malaria [54]. Knockdown of GSNOR was found to improve MPP + -induced toxicity in cellular models [25]. However, there were some studies showing that GSNOR knockout promoted the S-nitrosation of parkin and led to mitophagy defects [55,56], which caused a detrimental effect, instead of beneficial effect on the disease progression. ...
The S-nitrosoglutathione reductase (GSNOR) is a key denitrosylating enzyme that regulates protein S-nitrosation, a process which has been found to be involved in the pathogenesis of Parkinson's disease (PD). However, the physiological function of GSNOR in PD remains unknown. In a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model, we found that GSNOR expression was significantly increased and accompanied by autophagy mediated by MPTP-induced cyclin dependent kinase 5 (CDK5), behavioral dyskinesias and dopaminergic neuron loss. Whereas, knockout of GSNOR, or treatment with the GSNOR inhibitor N6022, alleviated MPTP-induced PD-like pathology and neurotoxicity. Mechanistically, deficiency of GSNOR inhibited MPTP-induced CDK5 kinase activity and CDK5-mediated autophagy by increasing S-nitrosation of CDK5 at Cys83. Our study indicated that GSNOR is a key regulator of CDK5 S-nitrosation and is actively involved in CDK5-mediated autophagy induced by MPTP.
... The concomitant occurrence of both these conditions drives cell senescence due to the accumulation of dysfunctional mitochondria [177]. Interestingly, mouse and human models of neuromuscular diseases (e.g., ALS and Duchenne muscular dystrophy) show GSNOR decrease and excessive SNO levels in the muscle and spinal cord [321][322][323], thereby confirming the detrimental role of excessive nitrosylation. ...
As mitochondria are vulnerable to oxidative damage and represent the main source of reactive oxygen species (ROS), they are considered key tuners of ROS metabolism and buffering, whose dysfunction can progressively impact neuronal networks and disease. Defects in DNA repair and DNA damage response (DDR) may also affect neuronal health and lead to neuropathology. A number of congenital DNA repair and DDR defective syndromes, indeed, show neurological phenotypes, and a growing body of evidence indicate that defects in the mechanisms that control genome stability in neurons acts as aging-related modifiers of common neurodegenerative diseases such as Alzheimer, Parkinson's, Huntington diseases and Amyotrophic Lateral Sclerosis.
In this review we elaborate on the established principles and recent concepts supporting the hypothesis that deficiencies in either DNA repair or DDR might contribute to neurodegeneration via mechanisms involving mitochondrial dysfunction/deranged metabolism.
... We previously demonstrated that GSNOR is expressed concomitantly with Nrf2 activation in cellular models of amyotrophic lateral sclerosis and plays a role in the protection against NO donors-induced cell death (Rizza et al, 2015). By matching this evidence with data shown above, we checked whether Nrf2 was responsible for GSNOR upregulation. ...
The denitrosylase S-nitrosoglutathione reductase (GSNOR) has been suggested to sustain mitochondrial removal by autophagy (mitophagy), functionally linking S-nitrosylation to cell senescence and aging. In this study, we provide evidence that GSNOR is induced at the translational level in response to hydrogen peroxide and mitochondrial ROS. The use of selective pharmacological inhi-bitors and siRNA demonstrates that GSNOR induction is an event downstream of the redox-mediated activation of ATM, which in turn phosphorylates and activates CHK2 and p53 as intermediate players of this signaling cascade. The modulation of ATM/GSNOR axis, or the expression of a redox-insensitive ATM mutant influences cell sensitivity to nitrosative and oxidative stress, impairs mitophagy and affects cell survival. Remarkably, this interplay modulates T-cell activation, supporting the conclusion that GSNOR is a key molecular effector of the antioxidant function of ATM and providing new clues to comprehend the pleiotropic effects of ATM in the context of immune function.
... To investigate the involvement of Nrf2 signaling in quercetin and xanthohumol-mediated neuroprotective effects, cells were co-treated with a specific Nrf2 inhibitor (5 µM trigonelline) along with quercetin or xanthohumol prior to CORT treatment. The dose of trigonelline was based on previous studies of Nrf2 inhibition in cellular models (Arlt et al., 2013;Rizza et al., 2015). ...
Background:
Polyphenols are phytochemicals that have been associated with therapeutic effects in stress-related disorders. Indeed, studies suggest that polyphenols exert significant neuroprotection against multiple neuronal injuries, including oxidative stress and neuroinflammation, but the mechanisms are unclear. Evidence indicates that polyphenol neuroprotection may be mediated by activation of Nrf2, a transcription factor associated with antioxidant and cell survival responses. On the other hand, in stress-linked disorders, Fkbp5 is a novel molecular target for treatment because of its capacity to regulate glucocorticoid receptor sensitivity. However, it is not clear the role Fkbp5 plays in polyphenol-mediated stress modulation. In this study, the neuroprotective effects and mechanisms of the naturally derived polyphenols xanthohumol and quercetin against cytotoxicity induced by corticosterone were investigated in primary cortical cells.
Methods:
Primary cortical cells containing both neurons and astrocytes were pre-incubated with different concentrations of quercetin and xanthohumol to examine the neuroprotective effects of polyphenols on cell viability, morphology, and gene expression following corticosterone insult.
Results:
Both polyphenols tested prevented the reduction of cell viability and alterations of neuronal/astrocytic numbers due to corticosterone exposure. Basal levels of Bdnf mRNA were also decreased after corticosterone insult; however, this was reversed by both polyphenol treatments. Interestingly, the Nrf2 inhibitor blocked xanthohumol but not quercetin-mediated neuroprotection. In contrast, we found that Fkbp5 expression is exclusively modulated by quercetin.
Conclusions:
These results suggest that naturally derived polyphenols protect cortical cells against corticosterone-induced cytotoxicity and enhance cell survival via modulation of the Nrf2 pathway and expression of Fkbp5.
... This phenomenon (termed SR Ca 2+ unloading) was further determined to result from a slow release of SR Ca 2+ via RyR. Of noteworth is that the Ca 2+ released through SR Ca 2+ unloading seems to be extruded from the cell, not resequestered by the SR or other organelles, and was demonstrated to occur with no increase in bulk Ca m (myoplasmic free Ca 2+ concentration) [81]. It was concluded that the net effect of this training-induced SR Ca 2+ unloading would be a lower SR Ca 2+ content and an increased subsarcolemmal Ca 2+ gradient, with no effect on bulk Ca m . ...
... L-type Ca 2+ channels are associated with endothelin response, so they may be associated with changes due to physical exercise. It is known that endurance training increases L-type Ca 2+ channel current density approximately twofold in all three arterial sizes, with no effect on voltage-dependent activation or inactivation characteristics [81]. Additionally, a significant correlation between treadmill endurance time and peak L type Ca 2+ current density was demonstrated in all three arterial sizes, supporting a direct association between endurance capacity and coronary smooth muscle L-type Ca 2+ current density. ...
... Rizza et al. observed that the silencing of GSNOR was shown to protect SH-SY5Y cells from toxins characteristic of Parkinson's disease. However, it was also observed that overexpression of this enzyme is a resistance factor for the treatment of amyotrophic lateral sclerosis [81]. It has also been shown in other investigations that the absence of GSNOR may be harmful and that this increase in S-nitrosylation that would cause its absence would be pathological. ...
The current book entitled Free Radicals, Antioxidants, and Diseases gives an idea of detecting free radicals in vivo by newer techniques and provides insights into the roles played by various antioxidants in combating diseases caused by oxidative stress. The chapters included in this volume showcase new investigation in this field by the research groups around the world.
... In particular, it has been found out that GSNOR activity plays a role in smooth muscle relaxation [37][38][39], neuronal signaling [29,40], cardiovascular system homeostasis [39,41], and inflammatory processes [30,42]. As a consequence, conditions in which GSNOR is lacking-and, thus, PSNOs are extremely increased-have been associated with a plethora of pathological states, such as increased susceptibility to bacterial infections [35], hypervascularization and resistance to stroke [43], spontaneous hepatocellular carcinoma (HCC) development [44,45], neuromuscular atrophy [46], neurodegenerative diseases [47], and aging [22]. ...
... Rizza et al. observed that the silencing of GSNOR was shown to protect SH-SY5Y cells from toxins characteristic of Parkinson's disease. However, it was also observed that overexpression of this enzyme is a resistance factor for the treatment of amyotrophic lateral sclerosis [81]. It has also been shown in other investigations that the absence of GSNOR may be harmful and that this increase in S-nitrosylation that would cause its absence would be pathological. ...
The most important clinical consequence of coronary disease is acute myocardial infarc-tion caused by an occlusion that limits the irrigation to the heart. Although the goldstandard treatment is to restore blood flow, this reperfusion causes inherent damage byincreasing the size of the infarcted area primarily through the opening of the mitochondrial permeability transition pore (MPTP). The cardioprotective effect of nitric oxide (NO)has been described to operate through S-nitrosylation of several important proteins in thecardiomyocytes such as the calcium channels RyR2 and the L-type Ca2+channel andmitochondrial proteins, including the MPTP. In this sense, an attractive strategy to pre-vent the ischemia-reperfusion damage is to increase the bioavailability of endogenous S-nitrosothiols. S-nitrosoglutathione reductase (GSNOR) is an enzyme involved in themetabolism of NO through denitrosylation, which would limit the cardioprotective effectof NO. Although inhibition of GSNOR has been studied in different organs, its effects onmyocardial reperfusion have not yet been fully elucidated. In this chapter, we review the pathophysiology underlying myocardial reperfusion injury and the opening of the MPTP along with the cardioprotective role of S-nitrosothiols and the potential role for GSNOR.
... Mouse primary cortical neurons (PCNs) were obtained from cerebral cortices of E15 C57BL/6 mice embryos, as previously reported [44]. Animals were used in accordance with both international guidelines and Italian law and always applying the 3Rs principle; expert and trained staff routinely monitored the health status of all animals enrolled in the study. ...
The exposure to extremely low-frequency magnetic fields (ELF-MFs) has been associated to increased risk of neurodegenerative diseases, although the underlying molecular mechanisms are still undefined. Since epigenetic modulation has been recently encountered among the key events leading to neuronal degeneration, we here aimed at assessing if the control of gene expression mediated by miRNAs, namely miRs-34, has any roles in driving neuronal cell response to 50-Hz (1 mT) magnetic field in vitro. We demonstrate that ELF-MFs drive an early reduction of the expression level of miR-34b and miR-34c in SH-SY5Y human neuroblastoma cells, as well as in mouse primary cortical neurons, by affecting the transcription of the common pri-miR-34. This modulation is not p53 dependent, but attributable to the hyper-methylation of the CpG island mapping within the miR-34b/c promoter. Incubation with N-acetyl-l-cysteine or glutathione ethyl-ester fails to restore miR-34b/c expression, suggesting that miRs-34 are not responsive to ELF-MF-induced oxidative stress. By contrast, we show that miRs-34 control reactive oxygen species production and affect mitochondrial oxidative stress triggered by ELF-MFs, likely by modulating mitochondria-related miR-34 targets identified by in silico analysis. We finally demonstrate that ELF-MFs alter the expression of the α-synuclein, which is specifically stimulated upon ELF-MFs exposure via both direct miR-34 targeting and oxidative stress. Altogether, our data highlight the potential of the ELF-MFs to tune redox homeostasis and epigenetic control of gene expression in vitro and shed light on the possible mechanism(s) producing detrimental effects and predisposing neurons to degeneration.
... The same consideration applies to nuclear factor (erythroid-derived 2)-like 2 (Nrf2), the master regulator of antioxidant response, which has been associated with, and differently modulated by, GSNOR depending on its expression. 86 As well as the other proteins so far mentioned, Nrf2 is often upregulated in cancer 87,88 and has been reported to confer resistance to genotoxic stresses mediated by anticancer drugs, which involves oxidative damage (to proteins and DNA) as one of the main mechanisms of action. 89 Nrf2 mediates the transcription of several antioxidant enzymes, as well as the adaptor protein p62, 90,91 whose accumulation has been associated with cancer. ...
Nitric oxide (NO) is a gaseous pleiotropic molecule that can both induce irreversible oxidative damages and modulate physiological signal transductions by transient protein modifications, the most important of which is the S-nitrosylation of cysteine residues. Being noxious and healthy, the role of NO in cancer is seemingly contradictory, as at low concentrations it mediates tumor growth and proliferation whereas at high concentrations it promotes apoptosis and cancer growth inhibition. However, it is becoming evident that when endogenously produced, such as upon inducible nitric oxide synthase (NOS) activation, NO acts to sustain tumorigenesis. Similarly, although less explored, defects and deficiency in the denitrosylating enzyme S-nitrosoglutathione reductase (GSNOR) have been associated with the development and malignancy of liver and breast cancers, suggesting a primary role for NO signaling—that is, S-nitrosylation, being deeply involved in neoplastic transformation and progression. In this review, we summarize past and recent evidence on the role of S-nitrosylation and GSNOR in different processes that contribute to cell transformation when deregulated, such as DNA damage repair, energetic metabolism, and cell death. We also outline possible S-nitrosylation–targeted proteins that might contribute to tumorigenesis, and, finally, we speculate on the role of GSNOR in regulating the oncogenic effects induced downstream.
... Several aging-related neurodegenerative diseases are, indeed, characterized by increased oxidative and nitrosative stress. In experimental models of neurodegenerations, we have recently reported that GSNOR modulation exerts different effects on neuronal viability according to the stimulus applied [73]. In particular we provided evidence that GSNOR ablation protects neuroblastoma cells from death induced by Parkinson's disease (PD)-inducing toxins (Fig. 7), presumably by activating NE-F2-related factor-2 (Nrf2)mediated antioxidant response [74], and by inducing the accumulation of GSNO. ...
... In particular we provided evidence that GSNOR ablation protects neuroblastoma cells from death induced by Parkinson's disease (PD)-inducing toxins (Fig. 7), presumably by activating NE-F2-related factor-2 (Nrf2)mediated antioxidant response [74], and by inducing the accumulation of GSNO. On the other hand, we found that GSNOR transcription is induced in conditions of nitrosative stress to preserve cell viability of in vitro models of familial amyotrophic lateral sclerosis (fALS) [73]. ...
S-nitrosylation is a major redox posttranslational modification involved in cell signaling. The steady state concentration of S-nitrosylated proteins depends on the balance between the relative ability to generate nitric oxide (NO) via NO synthase and to reduce nitrosothiols by denitrosylases. Numerous works have been published in last decades regarding the role of NO and S-nitrosylation in the regulation of protein structure and function, and in driving cellular activities in vertebrates. Notwithstanding an increasing number of observations indicates that impairment of denitrosylation equally affects cellular homeostasis, there is still no report providing comprehensive knowledge on the impact that denitrosylation has on maintaining correct physiological processes and organ activities. Among denitrosylases, S-nitrosoglutathione reductase (GSNOR) represents the prototype enzyme to disclose how denitrosylation plays a crucial role in tuning NO-bioactivity and how much it deeply impacts on cell homeostasis and human patho-physiology. In this review we attempt to illustrate the history of GSNOR discovery and provide the evidence so far reported in support of GSNOR implications in development and human disease.
