Fig 2 - uploaded by Jiming Kong
Content may be subject to copyright.
Rotenone and TTFA induce autophagy. HEK 293 and U87 cells were treated as described in Fig. 1. (A) The percentage of cells with AVOs (autophagosomes and autolysosomes) was determined by flow cytometry. Rates of AVO formation induced by rotenone (R) and TTFA (T) are indicated in (i) HEK 293 and (ii) U87 cells over a 72-hour time course. (iii) Effect of 3-MA (2.0 mM) on the formation of AVOs that were induced by rotenone or TTFA after a treatment of 48 hours in U87 and HEK 293 cells. P values less than 0.05 represent significant difference between conditions, as indicated. (B) Electron-microscopy pictures were taken of HEK 293 cells that were untreated (control) or treated with TTFA (0.5 mM) for 48 hours. Arrows represent double-membrane vacuoles in TTFA-treated cells (enlarged image). N represents the nucleus. (C) Formation of GFP-LC3-labeled vacuoles (dots). The percentage of cells with GFP- LC3-labeled vacuoles (dots) in (i) HEK 293 and (ii) U87 cells following rotenone or TFFA treatment over a 48-hour time course was determined. Error bars represent s.e. of three independent experiments. (iii) Representative pictures from three independent experiments of U87 cells treated with GFP alone; GFP-LC3 alone; GFP-LC3 and rotenone; and GFP-LC3, rotenone and 3-MA (2.0 mM) were captured by an Olympus microscope and coolsnap camera. The nucleolus was stained with DAPI (blue). (iv) HEK 293 and U87 cells were treated with rotenone or TTFA in the presence or absence of 3-MA (2.0 mM). (D) The conversion of LC3-I to LC3-II was determined in (i) HEK 293 and (ii) U87 cells treated with rotenone or TTFA for 6, 16 or 24 hours in the presence or absence of the lysosomal inhibitor NH 4 Cl (30 mM). Cells were lysed and western blotted for expression of LC3. Blots were stripped and re-
Source publication
Autophagy is a self-digestion process important for cell survival during starvation. It has also been described as a form of programmed cell death. Mitochondria are important regulators of autophagy-induced cell death and damaged mitochondria are often degraded by autophagosomes. Inhibition of the mitochondrial electron transport chain (mETC) induc...
Similar publications
In addition to adenosine triphosphate (ATP) production, mitochondria have been implicated in the regulation of several physiological responses in plants, such as programmed cell death (PCD) activation. Salicylic acid (SA) and reactive oxygen species (ROS) are essential signaling molecules involved in such physiological responses; however, the mecha...
Citations
... Finally, autophagy and/or mitophagy mechanisms act against cellular oxidative stress (Filomeni et al. 2015). Increased ROS production triggers autophagy to mediate clearing of ROS and oxidative damage (Chen et al. 2007). As mitochondria are major source of ROS, chronic impairment of any kind of mitochondrial function overproduces ROS and triggers a self-removal signal, or mitophagy, to eliminate the further oxidative environment (Schofield and Schafer 2021). ...
Sleep is an essential and evolutionarily conserved process that affects many biological functions that are also strongly regulated by cellular metabolism. The interdependence between sleep homeostasis and redox metabolism, in particular, is such that sleep deprivation causes redox metabolic imbalances in the form of over-production of ROS. Likewise (and vice versa), accumulation of ROS leads to greater sleep pressure. Thus, it is theorized that one of the functions of sleep is to act as the brain’s “antioxidant” at night by clearing oxidation built up from daily stress of the active day phase. In this review, we will highlight evidence linking sleep homeostasis and regulation to redox metabolism by discussing (1) the bipartite role that sleep–wake neuropeptides and hormones have in redox metabolism through comparing cross-species cellular and molecular mechanisms, (2) the evolutionarily metabolic changes that accompanied the development of sleep loss in cavefish, and finally, (3) some of the challenges of uncovering the cellular mechanism underpinning how ROS accumulation builds sleep pressure and cellularly, how this pressure is cleared.
... The understanding of mitochondrial dysfunction and its suspected role within psychiatric diseases have gained interest in recent years. Mitochondrial dysfunction can adversely influence neuronal function through direct and indirect pathways i.e., suboptimal energy production, altered kynurenine metabolism, redox stress, altered nitric-oxide synthase activity, increased apoptosis, and/or altered pyrimidine biosynthesis, fatty acid metabolism, calcium homeostasis and/or inflammation (Bustamante et al., 2007;Chen et al., 2007;Grace et al., 2006;Missiroli et al., 2020;Murphy, 2018;Wajner and Amaral, 2016;Wallace, 2007). The first sign, and arguably the largest body of evidence for the involvement of the mitochondria within these pathologies, are increases in oxidative stress markers observed in psychiatric pathologies (Salim, 2014;Smaga et al., 2015). ...
The Ndufs4 knockout (KO) mouse is a validated and robust preclinical model of mitochondrial diseases (specifically Leigh syndrome), that displays a narrow window of relative phenotypical normality, despite its inherent mitochondrial complex I dysfunction and severe phenotype. Preclinical observations related to psychiatric comorbidities that arise in patients with mitochondrial diseases and indeed in Leigh syndrome are, however, yet to be investigated in this model. Strengthening this narrative is the fact that major depression and bipolar disorder are known to present with deficits in mitochondrial function. We therefore screened the behavioural profile of male and female Ndufs4 KO mice (relative to heterozygous; HET and wildtype; WT mice) between postnatal days 28 and 35 for locomotor, depressive- and anxiety-like alterations and linked it with selected brain biomarkers, viz. serotonin, kynurenine, and redox status in brain areas relevant to psychiatric pathologies (i.e., prefrontal cortex, hippocampus, and striatum). The Ndufs4 KO mice initially displayed depressive-like behaviour in the tail suspension test on PND31 but not on PND35 in the forced swim test. In the mirror box test, increased risk resilience was observed. Serotonin levels of KO mice, compared to HET controls, were increased on PND36, together with increased tryptophan to serotonin and kynurenine turnover. Kynurenine to kynurenic acid turnover was however decreased, while reduced versus oxidized glutathione ratio (GSH/GSSG) was increased. When considering the comorbid psychiatric traits of patients with mitochondrial disorders, this work elaborates on the neuropsychiatric profile of the Ndufs KO mouse. Secondly, despite locomotor differences, Ndufs4 KO mice present with a behavioural profile not unlike rodent models of bipolar disorder, namely variable mood states and risk-taking behaviour. The model may elucidate the bio-energetic mechanisms underlying mood disorders, especially in the presence of mitochondrial disease. Studies are however required to further validate the model's translational relevance.
... Furthermore, the induction of autophagy was attenuated and the expression of autophagy-related LC3-I/II was diminished by NAC treatment in ILL-treated CRC cells, suggesting that ILL-induced autophagy is dependent on ROS production. Hydrogen peroxide (H 2 O 2 ) and 2-methoxyestradiol (2-ME) induced autophagic cell death in various cancer cells, which was inhibited by 3-MA or the deletion of Beclin-1 or Atg7 but not by pan caspase inhibitor Z-VAD [24,25]. Blocking autophagy did not reduce ROS generation, which is consistent with our results and places ROS upstream of autophagy. ...
Colorectal cancer (CRC) is one of the most common malignancies worldwide. Isolinderalactone (ILL), a sesquiterpene isolated from the root extract of Lindera aggregata, has been reported to exhibit anti–proliferative and anti–metastatic activities in various cancer cell lines. However, the mechanisms associated with its antitumor effects on CRC cells remain unclear. ILL treatment significantly suppressed proliferation and induced cell cycle G2/M arrest in CRC cells by inhibiting the expression of cyclin B, p–cdc2, and p–cdc25c and up–regulating the expression of p21. In addition, ILL induced mitochondria–associated apoptosis through the up–regulation of cleaved –caspase–9 and –3 expression. ILL induced autophagy by increasing the levels of LC3B in CRC cells, which was partially rescued by treatment with an autophagy inhibitor (chloroquine). Furthermore, ILL increases the accumulation of reactive oxygen species (ROS) and activates the MAPK pathway. Application of the ROS scavenger, N–acetyl cysteine (NAC), effectively inhibited ILL toxicity and reversed ILL–induced apoptosis, cell cycle arrest, autophagy, and ERK activation. Taken together, these results suggest that ILL induces G2/M phase arrest, apoptosis, and autophagy and activates the MAPK pathway via ROS–mediated signaling in human CRC cells.
... In our study, enzyme activity at temperatures 35 • C and higher was similar to the activity of enzymes measured below 15 • C, suggesting that although fish were surviving at these higher temperatures during the CT max study, they were accumulat-ing metabolic by-products. During this period, it is likely that largemouth bass were incurring a substantial amount of reactive oxygen species that ultimately leads to mitochondrial and cellular destruction (Yongqiang et al. 2007). These findings show a cellular cost to largemouth bass surviving at higher temperatures that matches what is seen in whole organism, long-term thermal tolerance studies. ...
Freshwater ecosystems are undergoing rapid thermal shifts, making it increasingly important to understand species-specific responses to these changes. Traditional techniques for determining a species’ thermal tolerance are often lethal and time consuming. Using the enzyme activity associated with the electron transport system (ETS; hereafter referred to as enzyme assay) may provide a non-lethal, rapid, and efficient alternative to traditional techniques for some species. We used largemouth bass Micropterus salmoides Lacepede, 1802 to test the efficacy of using an enzyme assay to determine thermal tolerance and respiratory exploitation in response to variable acclimation temperatures. Three tissue types were dissected from fish acclimated to 20, 25, or 30°C and used in ETS assays at temperatures ranging from 7.5-40°C. While there were significant differences among tissue types and acclimation temperatures, maximal enzyme activity occurred from 25.23-31.91°C. Fish lost equilibrium at 39-42°C in traditional CTmax trials, significantly higher than the upper optimum range determined via enzyme assays. The ratio of enzyme activity to measured whole organism respiration rate decreased with increasing water temperature, with the largest changes occurring at the upper optimum thermal range determined by enzyme assays. Our results indicate ETS analysis may prove useful for obtaining biologically relevant thermal tolerances.
... HEK-293 cells have also been used to demonstrate that, depending on its concentration, the neurotoxin ROT can induce sublethal and/or lethal effects. For instance, it has been shown that ROT (10 nM) might induce the cytosolic production of H 2 O 2 only in HEK-293 cells [90], whereas, at higher concentrations (e.g., 10 µM), it induces autophagy and apoptosis ( [45,91] and this work). Last but not least, HEK-293 cells have been used to ectopically express not only the human dopamine transporter (hDAT) to study, e.g., the toxic effect of MPTP [92], but also dopaminergic receptors, e.g., D1 [93] and/or D5 [94]. ...
Parkinson’s disease (PD) is a neurodegenerative disorder caused by the progressive loss of dopaminergic (DAergic) neurons in the substantia nigra and the intraneuronal presence of Lewy bodies (LBs), composed of aggregates of phosphorylated alpha-synuclein at residue Ser129 (p-Ser129α-Syn). Unfortunately, no curative treatment is available yet. To aggravate matters further, the etiopathogenesis of the disorder is still unresolved. However, the neurotoxin rotenone (ROT) has been implicated in PD. Therefore, it has been widely used to understand the molecular mechanism of neuronal cell death. In the present investigation, we show that ROT induces two convergent pathways in HEK-293 cells. First, ROT generates H2O2, which, in turn, either oxidizes the stress sensor protein DJ-Cys106-SH into DJ-1Cys106SO3 or induces the phosphorylation of the protein LRRK2 kinase at residue Ser395 (p-Ser395 LRRK2). Once active, the kinase phosphorylates α-Syn (at Ser129), induces the loss of mitochondrial membrane potential (ΔΨm), and triggers the production of cleaved caspase 3 (CC3), resulting in signs of apoptotic cell death. ROT also reduces glucocerebrosidase (GCase) activity concomitant with the accumulation of lysosomes and autophagolysosomes reflected by the increase in LC3-II (microtubule-associated protein 1A/1B-light chain 3-phosphatidylethanolamine conjugate II) markers in HEK-293 cells. Second, the exposure of HEK-293 LRRK2 knockout (KO) cells to ROT displays an almost-normal phenotype. Indeed, KO cells showed neither H2O2, DJ-1Cys106SO3, p-Ser395 LRRK2, p-Ser129α-Syn, nor CC3 but displayed high ΔΨm, reduced GCase activity, and the accumulation of lysosomes and autophagolysosomes. Similar observations are obtained when HEK-293 LRRK2 wild-type (WT) cells are exposed to the inhibitor GCase conduritol-β-epoxide (CBE). Taken together, these observations imply that the combined development of LRRK2 inhibitors and compounds for recovering GCase activity might be promising therapeutic agents for PD.
... Reportedly, mitochondrial OXPHOS inhibition leads to increased ROS production [20], which could also be the case with tamoxifen. We assessed whether tamoxifen increases ROS levels and compared differences between WT and TAM-R cells, and WT and Rho0 cells, by measuring the fluorescent intensity of CellROX indicating real-time cellular ROS production. ...
Background
Recently, we reported that tamoxifen-resistant (TAM-R) breast cancer cells are cross-resistant to irradiation. Here, we investigated the mechanisms associated with tamoxifen-induced radioresistance, aiming to prevent or reverse resistance and improve breast cancer treatment.
Methods
Wild-type ERα-positive MCF7 and ERα-negative MDA-MB-231 breast cancer cells and their TAM-R counterparts were analyzed for cellular metabolism using the Seahorse metabolic analyzer. Real-time ROS production, toxicity, and antioxidant capacity in response to H2O2, tamoxifen, and irradiation were determined. Tumor material from 28 breast cancer patients before and after short-term presurgical tamoxifen (ClinicalTrials.gov Identifier: NCT00738777, August 19, 2008) and cellular material was analyzed for NRF2 gene expression and immunohistochemistry. Re-sensitization of TAM-R cells to irradiation was established using pharmacological inhibition.
Results
TAM-R cells exhibited decreased oxygen consumption and increased glycolysis, suggesting mitochondrial dysfunction. However, this did not explain radioresistance, as cells without mitochondria (Rho-0) were actually more radiosensitive. Real-time measurement of ROS after tamoxifen and H2O2 exposure indicated lower ROS levels and toxicity in TAM-R cells. Consistently, higher antioxidant levels were found in TAM-R cells, providing protection from irradiation-induced ROS. NRF2, a main activator of the antioxidant response, was increased in TAM-R cells and in tumor tissue of patients treated with short-term presurgical tamoxifen. NRF2 inhibition re-sensitized TAM-R cells to irradiation.
Conclusion
Mechanisms underlying tamoxifen-induced radioresistance are linked to cellular adaptations to persistently increased ROS levels, leading to cells with chronically upregulated antioxidant capacity and glycolysis. Pharmacological inhibition of antioxidant responses re-sensitizes breast cancer cells to irradiation.
... Inhibition of the ETC leads to upregulation of ROS levels and induces autophagy, and continued disruption of the ETC leads to PCD. The addition of inhibitors of complex I and complex II to the mitochondrial ETC induces autophagy and death (Chen et al. 2007). Oxidative stress triggered by inhibitors of ETC such as antimycin A (AA) or methyl viologen (MV) induces high levels of plant autophagy, and this effect can be overcome by the exogenous addition of antioxidants (Deffieu et al. 2013). ...
... Inhibition of the ETC within mitochondria led to upregulation of ROS levels and induced autophagy, while persistent disruption of the ETC led to cellular PCD. For example, the addition of inhibitors of complex I and complex II in the ETC induced autophagy and death (Chen et al. 2007); oxidative stress triggered by ETC inhibitors such as antimycin A (AA) or methyl viologen (MV) induced high levels of plant autophagy. However, the mechanism of ETC in mitophagy remains unclear. ...
Mitochondria are crucial for the regulation of intracellular energy metabolism, biosynthesis, and cell survival. And studies have demonstrated the role of mitochondria in oxidative stress–induced autophagy in plants. Previous studies found that waterlogging stress can induce the opening of mitochondrial permeability transition pore (mPTP) and the release of cytochrome c in endosperm cells, which proved that mPTP plays an important role in the programmed cell death of endosperm cells under waterlogging stress. This study investigated the effects of the opening of mPTP and the inhibition of ETC on mitophagy in wheat roots under waterlogging stress. The results showed that autophagy related genes in the mitochondria of wheat root cells could respond to waterlogging stress; waterlogging stress led to the degradation of the characteristic proteins cytochrome c and COXII in the mitochondria of root cells. With the prolongation of waterlogging time, the protein degradation degree and the occurrence of mitophagy gradually increased. Under waterlogging stress, exogenous mPTP opening inhibitor CsA inhibited mitophagy in root cells and alleviated mitophagy induced by flooding stress, while exogenous mPTP opening inducer CCCP induced mitophagy in root cells; exogenous mPTP opening inducer CCCP induced mitophagy in root cells. The electron transfer chain inhibitor antimycin A induces mitophagy in wheat root cells and exacerbates mitochondrial degradation. In conclusion, waterlogging stress led to the degradation of mitochondrial characteristic proteins and the occurrence of mitophagy in wheat root cells, and the opening of mPTP and the inhibition of ETC induced the occurrence of mitophagy.
... Rotenone treatment induces mitochondrial homeostasis imbalance which might, initially, result in upregulated ROS levels. However, this, in turn, induces autophagy upregulation which, among a myriad of consequences, scavenges ROS [71]. Since we are monitoring chronic rotenone effects (following four and eight weeks of treatment), we, possibly, 'miss' ROS levels upregulation. ...
... Among the five mitochondrial respiration complexes, rotenone treatment for four weeks exerted a statistically significant effect only on hippocampal CoV and frontal-cortex CoIV protein levels, reducing the former and elevating the latter. The same rotenone regime also upregulated frontal-cortex LC3-II levels and LC3-II/p62 protein levels ration, indicative of autophagy upregulation as previously reported by us [32,103] and recently advised by Klionski et al. [80] for the Beclin 1/p62 ratio or its reciprocal, and previously reported by others in the cell lines HEK 293, U87 and HeLa [71]. All the above changes were eliminated by lithium, trehalose, and NAC + trehalose, and someby additional drugs used. ...
... Rotenone treatment induces mitochondrial homeostasis imbalance which might, initially, result in upregulated ROS levels. However, this, in turn, induces autophagy upregulation which, among a myriad of consequences, scavenges ROS [71]. Since we are monitoring chronic rotenone effects (following four and eight weeks of treatment), we, possibly, 'miss' ROS levels upregulation. ...
... Among the five mitochondrial respiration complexes, rotenone treatment for four weeks exerted a statistically significant effect only on hippocampal CoV and frontal-cortex CoIV protein levels, reducing the former and elevating the latter. The same rotenone regime also upregulated frontal-cortex LC3-II levels and LC3-II/p62 protein levels ration, indicative of autophagy upregulation as previously reported by us [32,103] and recently advised by Klionski et al. [80] for the Beclin 1/p62 ratio or its reciprocal, and previously reported by others in the cell lines HEK 293, U87 and HeLa [71]. All the above changes were eliminated by lithium, trehalose, and NAC + trehalose, and someby additional drugs used. ...
Bipolar-disorder’s pathophysiology and the mechanism by which medications exert their beneficial effect is yet unknown, but others’ and our data implicate patients’ brain mitochondrial-dysfunction and its amendment by mood-stabilizers. We recently designed a novel mouse bipolar-disorder-like model using chronic administration of a low-dose of the oxidative-phosphorylation complex I inhibitor, rotenone. Four and eight weeks rotenone treatment induced manic- and depressive-like behavior, respectively, accompanied by mood-related neurochemical changes. Here we aimed to investigate whether each of the autophagy-enhancers lithium (a mood-stabilizer), trehalose and resveratrol and/or each of the reactive oxygen species (ROS)-scavengers, resveratrol and N-acetylcystein and/or the combinations lithium+resveratrol or trehalose+N-acetylcystein, can ameliorate behavioral and neurochemical consequences of neuronal mild mitochondrial-dysfunction. We observed that lithium, trehalose and N-acetylcystein reversed rotenone-induced manic-like behavior as well as deviations in protein levels of mitochondrial complexes and the autophagy marker LC3-II. This raises the possibility that mild mitochondrial-dysfunction accompanied by impaired autophagy and a very mild increase in ROS levels are related to predisposition to manic-like behavior. On the other hand, although, as expected, most of the drugs tested eliminated the eight weeks rotenone-induced increase in protein levels of all hippocampal mitochondrial complexes, only lithium ubiquitously ameliorated the depressive-like behaviors. We cautiously deduce that aberrant autophagy and/or elevated ROS levels are not involved in predisposition to the depressive phase of bipolar-like behavior. Rather, that amending the depressive–like characteristics requires different mitochondria-related interventions. The latter might be antagonizing N-methyl-D-aspartate receptors (NMDARs), thus protecting from disruption of mitochondrial calcium homeostasis and its detrimental consequences. In conclusion, our findings suggest that by-and-large, among the autophagy-enhancers and ROS-scavengers tested, lithium is the most effective in counteracting rotenone-induced changes. Trehalose and N-acetylcystein may also be effective in attenuating manic-like behavior.
... ROT is a lipophilic insecticide that crosses the blood-brain barrier [38]. ROT accumulates in the mitochondria and mediates the inhibition of mitochondrial complex 1, thereby increasing ROS and pro-inflammatory factors [27,39,40]. Drugs that mitigate the ...
... ROT is a lipophilic insecticide that crosses the blood-brain barrier [38]. ROT accumulates in the mitochondria and mediates the inhibition of mitochondrial complex 1, thereby increasing ROS and pro-inflammatory factors [27,39,40]. Drugs that mitigate the burden of oxidative stress and inflammation may be potential therapeutic candidates for PD. ...
Parkinson’s disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta, resulting in motor deficits. The exact etiology of PD is currently unknown; however, the pathological hallmarks of PD include excessive production of reactive oxygen species, enhanced neuroinflammation, and overproduction of α-synuclein. Under normal physiological conditions, aggregated α-synuclein is degraded via the autophagy lysosomal pathway. However, impairment of the autophagy lysosomal pathway results in α-synuclein accumulation, thereby facilitating the pathogenesis of PD. Current medications only manage the symptoms, but are unable to delay, prevent, or cure the disease. Collectively, oxidative stress, inflammation, apoptosis, and autophagy play crucial roles in PD; therefore, there is an enormous interest in exploring novel bioactive agents of natural origin for their protective roles in PD. The present study evaluated the role of myrcene, a monoterpene, in preventing the loss of dopaminergic neurons in a rotenone (ROT)-induced rodent model of PD, and elucidated the underlying mechanisms. Myrcene was administered at a dose of 50 mg/kg, 30 min prior to the intraperitoneal injections of ROT (2.5 mg/kg). Administration of ROT caused a considerable loss of dopaminergic neurons, subsequent to a significant reduction in the antioxidant defense systems, increased lipid peroxidation, and activation of microglia and astrocytes, along with the production of pro-inflammatory cytokines (IL-6, TNF-α, IL-1β) and matrix metalloproteinase-9. Rotenone also resulted in impairment of the autophagy lysosomal pathway, as evidenced by increased expression of LC3, p62, and beclin-1 with decreased expression in the phosphorylation of mTOR protein. Collectively, these factors result in the loss of dopaminergic neurons. However, myrcene treatment has been observed to restore antioxidant defenses and attenuate the increase in concentrations of lipid peroxidation products, pro-inflammatory cytokines, diminished microglia, and astrocyte activation. Myrcene treatment also enhanced the phosphorylation of mTOR, reinstated neuronal homeostasis, restored autophagy-lysosomal degradation, and prevented the increased expression of α-synuclein following the rescue of dopaminergic neurons. Taken together, our study clearly revealed the mitigating effect of myrcene on dopaminergic neuronal loss, attributed to its potent antioxidant, anti-inflammatory, and anti-apoptotic properties, and favorable modulation of autophagic flux. This study suggests that myrcene may be a potential candidate for therapeutic benefits in PD.
