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NAD synthesis activity of Wld S is responsible for its protective effect on paraquat-induced cell death. (A) The NAD synthesis activity was required for the anti-apoptotic effect of Wld S. After transfection with the plasmids expressing EGFP, EGFP-Wld S (Wld S ), EGFP-Wld S H112A (H112A), or EGFP-Wld S F116S (F116S) for 24 h, the Hela cells were treated with paraquat for 24 h. Then the cells were stained with Hoechst 33342. Arrows indicate dead cells. Scale bar, 20 mm. (B) Quantification of the dead cells corresponding to (A). The percentage of dead cells was quantified in cells with green fluorescence. **p,0.01 compared with cells expressing EGFP, ##p,0.01 compared with cells expressing EGFP-Wld S , Student's ttest. (C) The expression of EGFP and EGFP-Wld S and its mutants were confirmed by western blot with anti-EGFP. Tubulin was measured as an internal control. (D) The decrease of NAD levels was delayed in Wld S MEFs treated with paraquat. Wild-type (WT) and Wld S MEFs were treated with 1 mM paraquat for the indicated times to assess the NAD levels. **p,0.01 versus WT with the same treatment, Student's t-test. doi:10.1371/journal.pone.0021770.g004
Source publication
Wld(S) is a fusion protein with NAD synthesis activity, and has been reported to protect axonal and synaptic compartments of neurons from various mechanical, genetic and chemical insults. However, whether Wld(S) can protect non-neuronal cells against toxic chemicals is largely unknown. Here we found that Wld(S) significantly reduced the cytotoxicit...
Contexts in source publication
Context 1
... to determine whether Wld S mitochondria are intrinsically more resistant to paraquat-induced injury, we isolated sufficient purified and functional mitochondria from the livers of wild-type and Wld S mice. After the isolated liver mitochondria were treated with the indicated concentrations of paraquat, we found that Wld S mitochondria showed no resistance to paraquat-induced disrup- tion of membrane potential when measured by JC-1 staining ( Figure S4B). In addition, we found Wld S protein also localized in the nuclei of primary mouse hepatocytes ( Figure S4A). ...
Context 2
... the isolated liver mitochondria were treated with the indicated concentrations of paraquat, we found that Wld S mitochondria showed no resistance to paraquat-induced disrup- tion of membrane potential when measured by JC-1 staining ( Figure S4B). In addition, we found Wld S protein also localized in the nuclei of primary mouse hepatocytes ( Figure S4A). Collective- ly, these data demonstrate that Wld S delays paraquat-induced mitochondrial injury in MEFs. ...
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... for the protective function against paraquat-induced cytotoxicity, we constructed plasmids expressing EGFP-fused Wld S or enzyme-dead Wld S with an H112A or F116S point mutation as previously described [7,34]. As shown in Figure 4A and Figure 4B, Wld S significantly protected Hela cells from paraquat-induced cell death, while enzyme-dead Wld S disrupted the protective effect of Wld S , and even exhibited an opposite effect to promote cell death. The expression of EGFP-fused Wld S and Wld S -H112A and Wld S -F116S protein were confirmed by western blot ( Figure 4C). ...
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... for the protective function against paraquat-induced cytotoxicity, we constructed plasmids expressing EGFP-fused Wld S or enzyme-dead Wld S with an H112A or F116S point mutation as previously described [7,34]. As shown in Figure 4A and Figure 4B, Wld S significantly protected Hela cells from paraquat-induced cell death, while enzyme-dead Wld S disrupted the protective effect of Wld S , and even exhibited an opposite effect to promote cell death. The expression of EGFP-fused Wld S and Wld S -H112A and Wld S -F116S protein were confirmed by western blot ( Figure 4C). ...
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... shown in Figure 4A and Figure 4B, Wld S significantly protected Hela cells from paraquat-induced cell death, while enzyme-dead Wld S disrupted the protective effect of Wld S , and even exhibited an opposite effect to promote cell death. The expression of EGFP-fused Wld S and Wld S -H112A and Wld S -F116S protein were confirmed by western blot ( Figure 4C). These results suggest that the protective effect of Wld S requires its NAD synthesis activity. ...
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... we examined whether Wld S could delay paraquat-induced NAD decrease. As shown in Figure 4D, intracellular NAD level was not significantly increased in Wld S MEFs at basal level. However, Wld S markedly delayed the NAD decline caused by paraquat ( Figure 4D), which is similar to its protective effect on cell viability. ...
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... shown in Figure 4D, intracellular NAD level was not significantly increased in Wld S MEFs at basal level. However, Wld S markedly delayed the NAD decline caused by paraquat ( Figure 4D), which is similar to its protective effect on cell viability. These results imply that Wld S could exert its protective effects via its NAD synthesis activity by attenuating NAD depletion after paraquat exposure. ...
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... were considered to be statistically significant when p,0.05. Figure S4 Liver mitochondria from Wld S mice are as sensitive as wild-type to paraquat-induced membrane potential disruption. (A) The nuclear localization of Wld S protein in primary mouse hepatocytes was measured by immuno- fluorescence using anti-Wld S antibody. ...
Citations
... The PQ dosages we used to treat the cells were used in previous in vitro studies. 30,62 However, in the present study, cells were not continuously incubated in reported concentrations of PQ. A short incubation time of 20 min was adopted, after which the PQ was replaced by normal culture medium so that we could observe uptake activities during this period of time and the effects of PQ intake to the cells. ...
Background:
Paraquat (PQ) is a pesticide, exposure to which has been associated with an increased risk of Parkinson's disease; however, PQ transport mechanisms in the brain are still unclear. Our previous studies indicated that the organic cation transporter 3 (OCT3) expressed on astrocytes could uptake PQ and protect the dopaminergic (DA) neurons from a higher level of extracellular PQ. At present, it is unknown how OCT3 levels are altered during chronic PQ exposure or aging, nor is it clear how the compensatory mechanisms are triggered by OCT3 deficiency. Dynamic related protein 1 (DRP1) was previously reported to ameliorate the loss of neurons during Parkinson's disease. Nowadays, mounting studies have revealed the functions of astrocyte DRP1, prompting us to hypothesize that DRP1 could regulate the PQ transport capacity of astrocytes.
Objectives:
The present study aimed to further explore PQ transport mechanisms in the nigrostriatal system and identify pathways involved in extracellular PQ clearance.
Methods:
Models of PQ-induced neurodegeneration were established by intraperitoneal (i.p.) injection of PQ in wild-type (WT) and organic cation transporter-3-deficient (Oct3-/-) mice. DRP1 knockdown was achieved by viral tools in vivo and small interfering RNA (siRNA) in vitro. Extracellular PQ was detected by in vivo microdialysis. In vitro transport assays were used to directly observe the functions of different transporters. PQ-induced neurotoxicity was evaluated by tyrosine hydroxylase immunohistochemistry, in vivo microdialysis for striatal DA and behavior tests. Western blotting analysis or immunofluorescence was used to evaluate the expression levels and locations of proteins in vitro or in vivo.
Results:
Older mice and those chronically exposed to PQ had a lower expression of brain OCT3 and, following exposure to a 10-mg/kg i.p. PQ2+ loading dose, a higher concentration of extracellular PQ. DRP1 levels were higher in astrocytes and neurons of WT and Oct3-/- mice after chronic exposure to PQ; this was supported by finding higher levels of DRP1 after PQ treatment of dopamine transporter-expressing neurons with and without OCT3 inhibition and in primary astrocytes of WT and Oct3-/- mice. Selective astrocyte DRP1 knockdown ameliorated the PQ2+-induced neurotoxicity in Oct3-/- mice but not in WT mice. GL261 astrocytes with siRNA-mediated DRP1 knockdown had a higher expression of alanine-serine-cysteine transporter 2 (ASCT2), and transport studies suggest that extracellular PQ was transported into astrocytes by ASCT2 when OCT3 was absent.
Discussion:
The present study mainly focused on the transport mechanisms of PQ between the dopaminergic neurons and astrocytes. Lower OCT3 levels were found in the older or chronically PQ-treated mice. Astrocytes with DRP1 inhibition (by viral tools or mitochondrial division inhibitor-1) had higher levels of ASCT2, which we hypothesize served as an alternative transporter to remove extracellular PQ when OCT3 was deficient. In summary, our data suggest that OCT3, ASCT2 located on astrocytes and the dopamine transporter located on DA terminals may function in a concerted manner to mediate striatal DA terminal damage in PQ-induced neurotoxicity. https://doi.org/10.1289/EHP9505.
... In addition, macronuclear genome of Tetrahymena thermophila is already sequenced, and its analysis revealed a higher degree of functional conservation with human genes than do other eukaryotic microbial models, such as yeasts (Eisen et al., 2006). Therefore, this protist is a good biological model to study environmental and human risks caused by xenobiotic exposures, representing an alternative to animal tests (Martín-Gonz alez et al., 1999;Miao et al., 2006;Yu et al., 2011). In the present research work, the PQ cytotoxicity, ROS generation, the mitochondrial membrane potential, cellular ultrastructural alterations and the gene expression level of several selected genes encoding relevant antioxidant enzymes, have been analysed on the ciliate-model T. thermophila. ...
... We have detected intense morphological alterations in these organelles. Likewise, ultrastructural changes and mitochondrial degeneration have been also reported, after PQ exposure, in microalgae (Bray et al., 1993), human lung cells (Wang et al., 1992) and non-neuronal mouse cells (Yu et al., 2011). Cell stress, including oxidative stress induced by environment or intracellular processes, can disturbance mitochondrial integrity and functioning. ...
•Tetrahymena thermophila is an eukaryotic microorganism very resistant to paraquat.•Superoxide production is significantly increased under paraquat exposure.•ROS induction is proportional to paraquat concentration, when toxicity does not generates a high cell mortality.•Mitochondria may be the main target of paraquat toxicity in T. thermophila.•An up-regulation of gene expression encoding critical antioxidant enzymes is involved in the cell response against this toxic pollutant.
... Therefore, the present study analyzed the NAD(H) and ATP content in SDI enhances SIRT1 protein expression and inhibits caspase 3 in the liver following I/R. As an important cellular function regulator and an NAD-dependent enzyme, SIRT1 deacetylase was quantified by an immunoblotting assay (16). As demonstrated in Fig. 4A and B, I/R markedly suppressed SIRT1 at the protein level while SDI alleviated this change. ...
Sorbitol dehydrogenase (SDH), a key enzyme of the polyol pathway, has recently been demonstrated to have an important role in mediating tissue ischemia/reperfusion (I/R) injury. The present study investigated how this enzyme may affect the ischemic liver and the mechanism underlying its effect. Firstly, C57BL/6 mice were subjected to oral administration of CP‑470,711 (5 mg/kg body weight/day for five days) and 70% hepatic I/R. Next the present study further investigated the changes in liver function, histology, inflammation, apoptosis and necrosis; the cytosolic adenosine triphosphate (ATP) and nictotinamide adenine dinucleotide [NAD(H)] contents and the protein level of caspase 3 and sirtuin 1 (SIRT1). The data demonstrated that sorbitol dehydrogenase inhibitor (SDI)‑administration significantly alleviated I/R‑induced liver injury, palliated histological changes and lowered the level of hepatocyte apoptosis and necrosis. In addition, SDI‑pretreatment in ischemic liver markedly maintained the cytosolic ATP and NAD(H) proportion, enhanced SIRT1 and suppressed the activation of caspase 3 at the protein level. The findings in the present study revealed that the flux through SDH may render the liver more vulnerable to I/R-induced injury and interventions targeting this enzyme may provide a novel adjunctive approach to protect from severe tissue injury following liver ischemia.
... This notion has been supported by several studies, in which NAD depletion following niacin deficiency or poly (ADP-ribose) polymerase overactivation aggravated tissue I/R injury, whereas the subsequent repletion of NAD or niacin markedly reversed this phenomenon [4,22,23]. The ameliorative effects of the restored NAD/NADH ratio have also been associated with the suppression of the HIF1 -TfR-Tf pathway and the enhanced activation of SIRT1 [5,14,24,25]. Until now, little attention has been given to the NAD/NADH ratio during hepatic I/R, especially in the fatty liver. Limited to the scale of this study, we did not explore all the courses discussed above, but the thread of NAD began to loom. ...
Background:
The polyol pathway, a bypass pathway of glucose metabolism initiated by aldose reductase (AR), has been shown to play an important role in mediating tissue ischemia/reperfusion (I/R) impairment recently. Here, we investigated how and why this pathway might affect the fatty liver following I/R.
Methods:
Two opposite models were created: mice with high-fat-diet-induced liver steatosis were treated with aldose reductase inhibition (ARI) and subsequent I/R; and AR-overexpressing L02 hepatocytes were sequentially subjected to steatosis and hypoxia/reoxygenation. We next investigated (a) the hepatic injuries, including liver function, histology, and hepatocytes apoptosis/necrosis; (b) the NAD(P)(H) contents, redox status, and mitochondrial function; and (c) the flux through the caspase-dependent apoptosis pathway.
Results:
AR-inhibition in vivo markedly attenuated the I/R-induced liver injuries, maintained the homeostasis of NAD(P)(H) contents and redox status, and suppressed the caspase-dependent apoptosis pathway. Correspondingly, AR overexpression in vitro presented the opposite effects.
Conclusion:
The flux through the polyol pathway may render steatotic liver greater vulnerability to I/R. Interventions targeting this pathway might provide a novel adjunctive approach to protect fatty liver from ischemia.
... Furthermore, Polm 2/2 mice were more resistant to acute treatment with paraquat ( Figure 4B), a potent inducer of oxidative stress that damages lung, liver, kidney and brain. This response is typically associated with mouse strains with an extended lifespan [21] and thought to be mediated by attenuation of the NAD depletion and reduced redox cycling and oxygen utilization associated with paraquat poisoning [22]; better functionality of Polm 2/2 liver could contribute to this increase resistance to paraquat. Analysis of microsatellite (MS) stability in liver cells revealed a mixed picture. ...
Polμ is an error-prone PolX polymerase that contributes to classical NHEJ DNA repair. Mice lacking Polμ (Polμ-/-) show altered hematopoiesis homeostasis and DSB repair and a more pronounced nucleolytic resection of some V(D)J junctions. We previously showed that Polμ-/- mice have increased learning capacity at old ages, suggesting delayed brain aging. Here we investigated the effect of Polμ-/- deficiency on liver aging. We found that old Polμ-/- mice (>20 month) have greater liver regenerative capacity compared with wt animals. Old Polμ-/- liver showed reduced genomic instability and increased apoptosis resistance. However, Polμ-/- mice did not show an extended life span and other organs (e.g., heart) aged normally. Our results suggest that Polμ deficiency activates transcriptional networks that reduce constitutive apoptosis, leading to enhanced liver repair at old age.
... Autophagy is a process that generates catabolic substrates for mitochondrial ATP production and allows cellular macromolecules to be recycled. Since we did not observe a difference in oxidative damage between wild-type and flcn-1(ok975) mutant, and since PQ is known to severely decrease ATP levels by inhibiting oxidative phosphorylation [73,74,75], we wondered if flcn-1 is mediating an increased resistance to energy stress by employing autophagy as a source of energy. To test this hypothesis, we measured ATP levels prior and after 13 hours of 10 mM PQ treatment. ...
Dysregulation of AMPK signaling has been implicated in many human diseases, which emphasizes the importance of characterizing AMPK regulators. The tumor suppressor FLCN, responsible for the Birt-Hogg Dubé renal neoplasia syndrome (BHD), is an AMPK-binding partner but the genetic and functional links between FLCN and AMPK have not been established. Strikingly, the majority of naturally occurring FLCN mutations predisposing to BHD are predicted to produce truncated proteins unable to bind AMPK, pointing to the critical role of this interaction in the tumor suppression mechanism. Here, we demonstrate that FLCN is an evolutionarily conserved negative regulator of AMPK. Using Caenorhabditis elegans and mammalian cells, we show that loss of FLCN results in constitutive activation of AMPK which induces autophagy, inhibits apoptosis, improves cellular bioenergetics, and confers resistance to energy-depleting stresses including oxidative stress, heat, anoxia, and serum deprivation. We further show that AMPK activation conferred by FLCN loss is independent of the cellular energy state suggesting that FLCN controls the AMPK energy sensing ability. Together, our data suggest that FLCN is an evolutionarily conserved regulator of AMPK signaling that may act as a tumor suppressor by negatively regulating AMPK function.
... Paraquat (N,N′-dimethyl-4,4′-bipyridinium dichloride) is a widely used herbicide that is toxic to kidney cells and functions to increase ROS levels in humans and animals through the inhibition of mitochondrial function. [32][33][34][35] Like that observed with Y 2 O 3 NPs, we found that the paraquat exposure induced a similar MTT response ( Figure 3C). The mechanism of the paraquat and the Y 2 O 3 NP cytotoxicity are similar but is not currently clear and will require further in-depth experimentation. ...
... To confirm these data biochemically, we next examined how the incubation with Y 2 O 3 NPs affected the ratio of Bcl-2 to Bax expression and caspase-3 fragmentation, given the role of these proteins in controlling the initiation and progression of the cellular apoptosis. 35 As expected from our flow cytometry data, we found that the incubation with the Y 2 O 3 NPs was associated with an increase in the ratio of Bax to Bcl-2 protein and caspase-3 fragmentation ( Figure 5A and 5B). Interestingly, we failed to observe any elevations in the caspase-3 fragmentation when the cells were incubated with 50 µg/mL of Y 2 O 3 NPs. ...
Background
The increased use of engineered nanoparticles (NPs) has caused new concerns about the potential exposure to biological systems and the potential risk that these materials may pose on human health. Here, we examined the effects of exposure to different concentrations (0–50 μg/mL) and incubation times (10 hours, 24 hours, or 48 hours) of yttrium oxide (Y2O3) NPs on human embryonic kidney (HEK293) cells. Changes in cellular morphology, cell viability, cell membrane integrity, reactive oxygen species levels, mitochondrial membrane potential, cell death (apoptosis and necrosis), and the DNA damage after NP exposure were compared to the effects seen following incubation with paraquat, a known toxicant.
Results
The 24-hour inhibitory concentration 50 (IC50) of Y2O3 NPs (41±5 nm in size) in the HEK293 cells was found to be 108 μg/mL. Incubation with Y2O3 NPs (12.25–50 μg/mL) increased the ratio of Bax/Bcl-2, caspase-3 expression and promoted apoptotic- and necrotic-mediated cell death in both a concentration and a time-dependent manner. Decreases in cell survivability were associated with elevations in cellular reactive oxygen species levels, increased mitochondrial membrane permeability, and evidence of DNA damage, which were consistent with the possibility that mitochondria impairment may play an important role in the cytotoxic response.
Conclusion
These data demonstrate that the Y2O3 NP exposure is associated with increased cellular apoptosis and necrosis in cultured HEK293 cells.
Background
Myofascial Pain Syndrome (MPS) is a common, overlooked, and underdiagnosed condition and has significant burden. MPS is often dismissed by clinicians while patients remain in pain for years. MPS can evolve into fibromyalgia, however, effective treatments for both are lacking due to absence of a clear mechanism. Many studies focus on central sensitization. Therefore, the purpose of this scoping review is to systematically search cross-disciplinary empirical studies of MPS, focusing on mechanical aspects, and suggest an organic mechanism explaining how it might evolve into fibromyalgia. Hopefully, it will advance our understanding of this disease.
Methods
Systematically searched multiple phrases in MEDLINE, EMBASE, COCHRANE, PEDro, and medRxiv, majority with no time limit. Inclusion/exclusion based on title and abstract, then full text inspection. Additional literature added on relevant side topics. Review follows PRISMA-ScR guidelines. PROSPERO yet to adapt registration for scoping reviews.
Findings
799 records included. Fascia can adapt to various states by reversibly changing biomechanical and physical properties. Trigger points, tension, and pain are a hallmark of MPS. Myofibroblasts play a role in sustained myofascial tension. Tension can propagate in fascia, possibly supporting a tensegrity framework. Movement and mechanical interventions treat and prevent MPS, while living sedentarily predisposes to MPS and recurrence.
Conclusions
MPS can be seen as a pathological state of imbalance in a natural process; manifesting from the inherent properties of the fascia, triggered by a disrupted biomechanical interplay. MPS might evolve into fibromyalgia through deranged myofibroblasts in connective tissue (“fascial armoring”). Movement is an underemployed requisite in modern lifestyle. Lifestyle is linked to pain and suffering. The mechanism of needling is suggested to be more mechanical than currently thought. A “global percutaneous needle fasciotomy” that respects tensegrity principles may treat MPS/fibromyalgia more effectively. “Functional-somatic syndromes” can be seen as one entity (myofibroblast-generated-tensegrity-tension), sharing a common rheuma-psycho-neurological mechanism.
Mitochondrial failure has long been associated with programmed axon death (Wallerian degeneration, WD), a widespread and potentially preventable mechanism of axon degeneration. While early findings in axotomised axons indicated that mitochondria are involved during the execution steps of this pathway, recent studies suggest that in addition, mitochondrial dysfunction can initiate programmed axon death without physical injury. As mitochondrial dysfunction is associated with disorders involving early axon loss, including Parkinson’s disease, peripheral neuropathies, and multiple sclerosis, the findings that programmed axon death is activated by mitochondrial impairment could indicate the involvement of druggable mechanisms whose disruption may protect axons in such diseases. Here, we review the latest developments linking mitochondrial dysfunction to programmed axon death and discuss their implications for injury and disease.
An approach for enzyme therapeutics is elaborated with cell-implanted nanoreactors that are based on multiple enzymes encapsulated in hollow silica nanospheres (HSNs). The synthesis of HSNs is carried out by silica sol–gel templating of water-in-oil microemulsions so that polyethyleneimine (PEI) modified enzymes in aqueous phase are encapsulated inside the HSNs. PEI-grafted superoxide dismutase (PEI-SOD) and catalase (PEI-CAT) encapsulated in HSNs are prepared with quantitative control of the enzyme loadings. Excellent activities of superoxide dismutation by PEI-SOD@HSN are found and transformation of H2O2 to water by PEI-CAT@HSN. When PEI-SOD and PEI-CAT are co-encapsulated, cascade transformation of superoxide through hydrogen peroxide to water was facile. Substantial fractions of HSNs exhibit endosome escape to cytosol after their delivery to cells. The production of downstream reactive oxygen species (ROS) and COX-2/p-p38 expression show that co-encapsulated SOD/CAT inside the HSNs renders the highest cell protection against the toxicant N,N′-dimethyl-4,4′-bipyridinium dichloride (paraquat). The rapid cell uptake and strong detoxification effect on superoxide radicals by the SOD/CAT-encapsulated hollow mesoporous silica nanoparticles demonstrate the general concept of implanting catalytic nanoreactors in biological cells with designed functions.
