Are mitochondrial reactive oxygen species required for autophagy?

Reactive oxygen species (ROS) are said to participate in the autophagy signaling. Supporting evidence is obscured by interference of autophagy and apoptosis, whereby the latter heavily relies on ROS signaling. To dissect autophagy from apoptosis we knocked down expression of cytochrome c, the key component of mitochondria-dependent apoptosis, in HeLa cells using shRNA. In cytochrome c deficient HeLa1.2 cells, electron transport was compromised due to the lack of electron shuttle between mitochondrial respiratory complexes III and IV. A rapid and robust LC3-I/II conversion and mitochondria degradation were observed in HeLa1.2 cells treated with staurosporine (STS). Neither generation of superoxide nor accumulation of H₂O₂ was detected in STS-treated HeLa1.2 cells. A membrane permeable antioxidant, PEG-SOD, plus catalase exerted no effect on STS-induced LC3-I/II conversion and mitochondria degradation. Further, STS caused autophagy in mitochondria DNA-deficient ρ° HeLa1.2 cells in which both electron transport and ROS generation were completely disrupted. Counter to the widespread view, we conclude that mitochondrial ROS are not required for the induction of autophagy.     

Constitutive Reactive Oxygen Species Generation from Autophagosome/Lysosome in Neuronal Oxidative Toxicity

Reactive oxygen species (ROS) are involved in several cell death processes, including cerebral ischemic injury. We found that glutamate-induced ROS accumulation and the associated cell death in mouse hippocampal cell lines were delayed by pharmacological inhibition of autophagy or lysosomal activity. Glutamate, however, did not stimulate autophagy, which was assessed by a protein marker, LC3, and neither changes in organization of mitochondria nor lysosomal membrane permeabilization were observed. Fluorescent analyses by a redox probe PF-H₂TMRos revealed that autophagosomes and/or lysosomes are the major sites for basal ROS generation in addition to mitochondria. Treatments with inhibitors for autophagy and lysosomes decreased their basal ROS production and caused a burst of mitochondrial ROS to be delayed. On the other hand, attenuation of mitochondrial activity by serum depletion or by high cell density culture resulted in the loss of both constitutive ROS production and an ROS burst in mitochondria. Thus, constitutive ROS production within mitochondria and lysosomes enables cells to be susceptible to glutamate-induced oxidative cytotoxicity. Likewise, inhibitors for autophagy and lysosomes reduced neural cell death in an ischemia model in rats. We suggest that cell injury during periods of ischemia is regulated by ROS-generating activity in autophagosomes and/or lysosomes as well as in mitochondria.     

Severe mitochondrial damage associated with low-dose radiation sensitivity in ATM- and NBS1-deficient cells

Low-dose radiation risks remain unclear owing to a lack of sufficient studies. We previously reported that low-dose, long-term fractionated radiation (FR) with 0.01 or 0.05 Gy/fraction for 31 d inflicts oxidative stress in human fibroblasts due to excess levels of mitochondrial reactive oxygen species (ROS). To identify the small effects of low-dose radiation, we investigated how mitochondria respond to low-dose radiation in radiosensitive human ataxia telangiectasia mutated (ATM)- and Nijmegen breakage syndrome (NBS)1-deficient cell lines compared with corresponding cell lines expressing ATM and NBS1. Consistent with previous results in normal fibroblasts, low-dose, long-term FR increased mitochondrial mass and caused accumulation of mitochondrial ROS in ATM- and NBS1-complemented cell lines. Excess mitochondrial ROS resulted in mitochondrial damage that was in turn recognized by Parkin, leading to mitochondrial autophagy (mitophagy). In contrast, ATM- and NBS1-deficient cells showed defective induction of mitophagy after low-dose, long-term FR, leading to accumulation of abnormal mitochondria; this was determined by mitochondrial fragmentation and decreased mitochondrial membrane potential. Consequently, apoptosis was induced in ATM- and NBS1-deficient cells after low-dose, long-term FR. Antioxidant N-acetyl-L-cysteine was effective as a radioprotective agent against mitochondrial damage induced by low-dose, long-term FR among all cell lines, including radiosensitive cell lines. In conclusion, we demonstrated that mitochondria are target organelles of low-dose radiation. Mitochondrial response influences radiation sensitivity in human cells. Our findings provide new insights into cancer risk estimation associated with low-dose radiation exposure.     

Regulation by mitophagy

Eukaryotes employ elaborate mitochondrial quality control to maintain the function of the power-generating organelle. Mitochondrial quality control is particularly important for the maintenance of neural and muscular tissues. Mitophagy is specialized version of the autophagy pathway. Mitophagy delivers damaged mitochondria to lysosomes for degradation. Recently, a series of elegant studies have demonstrated that two Parkinson's disease-associated genes PINK1 and parkin are involved in the maintenance of healthy mitochondria as mitophagy. Parkin in co-operation with PINK1 specifically recognizes damaged mitochondria with reduced mitochondrial membrane potential (Δψm), rapidly isolates them from the mitochondrial network and eliminates them through the ubiquitin–proteasome and autophagy pathways. Here we introduce and review recent studies that contribute to understanding the molecular mechanisms of mitophagy such as PINK1 and Parkin-mediated mitochondrial regulation. We also discuss how defects in the PINK1–Parkin pathway may cause neurodegeneration in Parkinson's disease.     

Mitochondrial Ubiquitin Ligase MITOL Ubiquitinates Mutant SOD1 and Attenuates Mutant SOD1-induced Reactive Oxygen Species Generation

We have previously identified a novel mitochondrial ubiquitin ligase, MITOL, which is localized in the mitochondrial outer membrane and is involved in the control of mitochondrial dynamics. In this study, we examined whether MITOL eliminates misfolded proteins localized to mitochondria. Mutant superoxide dismutase1 (mSOD1), one of misfolded proteins, has been shown to localize in mitochondria and induce mitochondrial dysfunction, possibly involving in the onset and progression of amyotrophic lateral sclerosis. We found that in the mitochondria, MITOL interacted with and ubiquitinated mSOD1 but not wild-type SOD1. In vitro ubiquitination assay revealed that MITOL directly ubiquitinates mSOD1. Cycloheximide-chase assay in the Neuro2a cells indicated that MITOL overexpression promoted mSOD1 degradation and suppressed both the mitochondrial accumulation of mSOD1 and mSOD1-induced reactive oxygen species (ROS) generation. Conversely, the overexpression of MITOL CS mutant and MITOL knockdown by specific siRNAs resulted in increased accumulation of mSOD1 in mitochondria, which enhanced mSOD1-induced ROS generation and cell death. Thus, our findings indicate that MITOL plays a protective role against mitochondrial dysfunction caused by the mitochondrial accumulation of mSOD1 via the ubiquitin–proteasome pathway.     

Effect of IGF-1 on the balance between autophagy of dysfunctional mitochondria and apoptosis

Mutations in mitochondrial DNA (mtDNA) cause excessive production of mitochondrial reactive oxygen species (ROS) and shorten animal life span. We examined the mechanisms responsible for removal of mitochondria with deleterious mtDNA mutations by autophagy. Incubation of primary cells and cell lines in the absence of serum promotes autophagy of mitochondria with deleterious mtDNA mutations but spares their normal counterparts. The effect of serum withdrawal on the autophagy of dysfunctional mitochondria is prevented by the addition of IGF-1. As a result of the elimination of mitochondria with deleterious mutations, excessive ROS production, characteristic of dysfunctional mitochondria, is greatly reduced. Mitochondrial autophagy shares a common mechanism with mitochondrial-induced cell apoptosis, including mitochondrial transition pore formation and increased ROS production.     

Reactive oxygen species induced by proteasome inhibition in neuronal cells mediate mitochondrial dysfunction and a caspase-independent cell death

While increasing evidence shows that proteasome inhibition triggers oxidative damage, mitochondrial dysfunction and death in neuronal cells, the regulatory relationship among these events is unclear. Using mouse neuronal cells we show that the cytotoxicity induced by mild (0.25 μM) and potent (5.0 μM) doses of the proteasome inhibitor, N-Benzyloxycarbonyl-Ile-Glu (O-t-butyl)-Ala-leucinal, (PSI) involved a dose-dependent increase in caspase activation, overproduction of reactive oxygen species (ROS) and a mitochondrial dysfunction manifested by the translocation of the proapoptotic protein, Bax, from the cytoplasm to the mitochondria, membrane depolarization and the release of cytochrome c and the apoptosis inducing factor (AIF) from mitochondria to the cytoplasm and nucleus, respectively. Whereas caspase or Bax inhibition failed to prevent mitochondrial membrane depolarization and neuronal cell death, pretreatments with the antioxidant N-acetyl-l-cysteine (NAC) or overexpression of the antiapoptotic protein Bcl-xL abrogated these events in cells exposed to mild levels of PSI. These findings implicated ROS as a mediator of PSI-induced cytotoxicity. However, depletions in glutathione and Bcl-xL with potent proteasome inhibition exacerbated this response whereupon survival required the cooperative protection of NAC with Bcl-xL overexpression. Collectively, ROS induced by proteasome inhibition mediates a mitochondrial dysfunction in neuronal cells that culminates in death through caspase- and Bax-independent mechanisms. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Depletion of NBR1 in urothelial carcinoma cells enhances rapamycin-induced apoptosis through impaired autophagy and mitochondrial dysfunction

Rapamycin is well-recognized in the clinical therapeutic intervention for patients with cancer by specifically targeting mammalian target of rapamycin (mTOR) kinase. Rapamycin regulates general autophagy to clear damaged cells. Previously, we identified increased expression of messenger RNA levels of NBR1 (the neighbor of BRCA1 gene; autophagy cargo receptor) in human urothelial cancer (URCa) cells, which were not exhibited in response to rapamycin treatment for cell growth inhibition. Autophagy plays an important role in cellular physiology and offers protection against chemotherapeutic agents as an adaptive response required for maintaining cellular energy. Here, we hypothesized that loss of NBR1 sensitizes human URCa cells to growth inhibition induced by rapamycin treatment, leading to interruption of protective autophagic activation. Also, the potential role of mitochondria in regulating autophagy was tested to clarify the mechanism by which rapamycin induces apoptosis in NBR1-knockdown URCa cells. NBR1-knockdown URCa cells exhibited enhanced sensitivity to rapamycin associated with the suppression of autophagosomal elongation and mitochondrial defects. Loss of NBR1 expression altered the cellular responses to rapamycin treatment, resulting in impaired ATP homeostasis and an increase in reactive oxygen species (ROS). Although rapamycin treatment-induced autophagy by adenosine monophosphate-activated protein kinase (AMPK) phosphorylation in NBR1-knockdown cells, it did not process the conjugated form of LC3B-II after activation by unc-51 like autophagy-activating kinase 1 (ULK1). NBR1-knockdown URCa cells exhibited rather profound mitochondrial dysfunctions in response to rapamycin treatment as evidenced by Δψm collapse, ATP depletion, ROS accumulation, and apoptosis activation. Therefore, our findings provide a rationale for rapamycin treatment of NBR1-knockdown human urothelial cancer through the regulation of autophagy and mitochondrial dysfunction by regulating the AMPK/mTOR signaling pathway, indicating that NBR1 can be a potential therapeutic target of human urothelial cancer.     

The Role of Autophagy in Mitochondria Maintenance: Characterization of Mitochondrial Functions in Autophagy-Deficient S. cerevisiae Strains

Autophagy is a lysosome-dependent cellular degradation process. Organisms bearing deletions of the essential autophagy genes exhibit various pathological conditions, including cancer in mammals and shortened life span in C. elegans. The direct cause for these phenotypes is not clear. Here we used yeast as a model system to characterize the cellular consequence of ATG (autophagy-related) gene deletions. We found that the atg mutant strains, atg1?, atg6?, atg8? and atg12?, showed defects related to mitochondrial biology. These strains were unable to degrade mitochondria in stationary culture. In non-fermentable medium, which requires mitochondrial oxidative phosphorylation for survival, these atg strains showed a growth defect with an increased cell population at the G₁ phase of the cell cycle. The cells had lower oxygen consumption rates and reduced mitochondrial electron transport chain activities. Under these growth conditions, the atg strains had lower mitochondrial membrane potential. In addition, these mutants generated higher levels of reactive oxygen species (ROS) and they were prone to accumulate dysfunctional mitochondria. This study clearly indicates that an autophagy defect has a functional impact on various aspects of mitochondrial functions and suggests a critical role of autophagy in mitochondria maintenance.     

Mitochondrial accumulation under oxidative stress is due to defects in autophagy

Mitochondrial dynamics maintains normal mitochondrial function by degrading damaged mitochondria and generating newborn mitochondria. The accumulation of damaged mitochondria influences the intracellular environment by promoting mitochondrial dysfunction, and thus initiating a vicious cycle. Oxidative stress induces mitochondrial malfunction, which is involved in many cardiovascular diseases. However, the mechanism of mitochondrial accumulation in cardiac myoblasts remains unclear. We observed mitochondrial dysfunction and an increase in mitochondrial mass under the oxidative conditions produced by tert‐butyl hydroperoxide (tBHP) in cardiac myoblast H9c2 cells. However, in contrast to the increase in mitochondrial mass, mitochondrial DNA (mtDNA) decreased, suggesting that enhanced mitochondrial biogenesis may be not the primary cause of the mitochondrial accumulation. Therefore, we investigated changes in a number of proteins involved in autophagy. Beclin1, Atg12–Atg5 conjugate, Atg7 contents decreased but LC3‐II accumulated in tBHP‐treated H9c2 cells. Moreover, the capacity for acid hydrolysis decreased in H9c2 cells. We also demonstrated a decrease in DJ‐1 protein under the oxidative conditions that deregulate mitochondrial dynamics. These results reveal that autophagy became defective under oxidative stress. We therefore suggest that defects in autophagy mediate mitochondrial accumulation under these conditions. J. Cell. Biochem. 114: 212–219, 2012. © 2012 Wiley Periodicals, Inc.     

Suppression of basal autophagy reduces lung cancer cell proliferation and enhances caspase-dependent and -independent apoptosis by stimulating ROS formation

Autophagy is a catabolic process involved in the turnover of organelles and macromolecules which, depending on conditions, may lead to cell death or preserve cell survival. We found that some lung cancer cell lines and tumor samples are characterized by increased levels of lipidated LC3. Inhibition of autophagy sensitized non-small cell lung carcinoma (NSCLC) cells to cisplatin-induced apoptosis; however, such response was attenuated in cells treated with etoposide. Inhibition of autophagy stimulated ROS formation and treatment with cisplatin had a synergistic effect on ROS accumulation. Using genetically encoded hydrogen peroxide probes directed to intracellular compartments we found that autophagy inhibition facilitated formation of hydrogen peroxide in the cytosol and mitochondria of cisplatin-treated cells. The enhancement of cell death under conditions of inhibited autophagy was partially dependent on caspases, however, antioxidant NAC or hydroxyl radical scavengers, but not the scavengers of superoxide or a MnSOD mimetic, reduced the release of cytochrome c and abolished the sensitization of the cells to cisplatin-induced apoptosis. Such inhibition of ROS prevented the processing and release of AIF (apoptosis-inducing factor) and HTRA2 from mitochondria. Furthermore, suppression of autophagy in NSCLC cells with active basal autophagy reduced their proliferation without significant effect on the cell-cycle distribution. Inhibition of cell proliferation delayed accumulation of cells in the S phase upon treatment with etoposide that could attenuate the execution stage of etoposide-induced apoptosis. These findings suggest that autophagy suppression leads to inhibition of NSCLC cell proliferation and sensitizes them to cisplatin-induced caspase-dependent and -independent apoptosis by stimulation of ROS formation.     

Autophagy protects auditory hair cells against neomycin-induced damage

Aminoglycosides are toxic to sensory hair cells (HCs). Macroautophagy/autophagy is an essential and highly conserved self-digestion pathway that plays important roles in the maintenance of cellular function and viability under stress. However, the role of autophagy in aminoglycoside-induced HC injury is unknown. Here, we first found that autophagy activity was significantly increased, including enhanced autophagosome-lysosome fusion, in both cochlear HCs and HEI-OC-1 cells after neomycin or gentamicin injury, suggesting that autophagy might be correlated with aminoglycoside-induced cell death. We then used rapamycin, an autophagy activator, to increase the autophagy activity and found that the ROS levels, apoptosis, and cell death were significantly decreased after neomycin or gentamicin injury. In contrast, treatment with the autophagy inhibitor 3-methyladenine (3-MA) or knockdown of autophagy-related (ATG) proteins resulted in reduced autophagy activity and significantly increased ROS levels, apoptosis, and cell death after neomycin or gentamicin injury. Finally, after neomycin injury, the antioxidant N-acetylcysteine could successfully prevent the increased apoptosis and HC loss induced by 3-MA treatment or ATG knockdown, suggesting that autophagy protects against neomycin-induced HC damage by inhibiting oxidative stress. We also found that the dysfunctional mitochondria were not eliminated by selective autophagy (mitophagy) in HEI-OC-1 cells after neomycin treatment, suggesting that autophagy might not directly target the damaged mitochondria for degradation. This study demonstrates that moderate ROS levels can promote autophagy to recycle damaged cellular constituents and maintain cellular homeostasis, while the induction of autophagy can inhibit apoptosis and protect the HCs by suppressing ROS accumulation after aminoglycoside injury.     

Giant mitochondria do not fuse and exchange their contents with normal mitochondria

Giant mitochondria accumulate within aged or diseased postmitotic cells as a consequence of insufficient autophagy, which is normally responsible for mitochondrial degradation. We report that giant mitochondria accumulating in cultured rat myoblasts due to inhibition of autophagy have low inner membrane potential and do not fuse with each other or with normal mitochondria. In addition to the low inner mitochondrial membrane potential in giant mitochondria, the quantity of the OPA1 mitochondrial fusion protein in these mitochondria was low, but the abundance of mitofusin-2 (Mfn2) remained unchanged. The combination of these factors may explain the lack of mitochondrial fusion in giant mitochondria and imply that the dysfunctional giant mitochondria cannot restore their function by fusing and exchanging their contents with fully functional mitochondria. These findings have important implications for understanding the mechanisms of accumulation of age-related mitochondrial damage in postmitotic cells.     

ROS, mitochondria and the regulation of autophagy

Accumulation of reactive oxygen species (ROS) is an oxidative stress to which cells respond by activating various defense mechanisms or, finally, by dying. At low levels, however, ROS act as signaling molecules in various intracellular processes. Autophagy, a process by which eukaryotic cells degrade and recycle macromolecules and organelles, has an important role in the cellular response to oxidative stress. Here, we review recent reports suggesting a regulatory role for ROS of mitochondrial origin as signaling molecules in autophagy, leading, under different circumstances, to either survival or cell death. We then discuss the relationship between mitochondria and autophagosomes and propose that mitochondria have an essential role in autophagosome biogenesis.     

Thyroid hormone induction of mitochondrial activity is coupled to mitophagy via ROS-AMPK-ULK1 signaling

Currently, there is limited understanding about hormonal regulation of mitochondrial turnover. Thyroid hormone (T₃) increases oxidative phosphorylation (OXPHOS), which generates reactive oxygen species (ROS) that damage mitochondria. However, the mechanism for maintenance of mitochondrial activity and quality control by this hormone is not known. Here, we used both in vitro and in vivo hepatic cell models to demonstrate that induction of mitophagy by T₃ is coupled to oxidative phosphorylation and ROS production. We show that T₃ induction of ROS activates CAMKK2 (calcium/calmodulin-dependent protein kinase kinase 2, β) mediated phosphorylation of PRKAA1/AMPK (5′ AMP-activated protein kinase), which in turn phosphorylates ULK1 (unc-51 like autophagy activating kinase 1) leading to its mitochondrial recruitment and initiation of mitophagy. Furthermore, loss of ULK1 in T₃-treated cells impairs both mitophagy as well as OXPHOS without affecting T₃ induced general autophagy/lipophagy. These findings demonstrate a novel ROS-AMPK-ULK1 mechanism that couples T₃-induced mitochondrial turnover with activity, wherein mitophagy is necessary not only for removing damaged mitochondria but also for sustaining efficient OXPHOS.     

The axis of mTOR-mitochondria-ROS and stemness of the hematopoietic stem cells

It has been hypothesized that adult hematopoietic stem cells (HSCs) need to remain quiescent to retain their long-term self-renewal activity and multipotency. However, it is still unclear how lack of quiescence is detrimental to HSC. We identified that the mTOR pathway is the key to HSCs quiescence. mTOR overactivation caused increased mitochondrial biogenesis and accumulation of much higher level of reactive oxygen species (ROS). Removal of ROS rescued HSC defects associated with hyperactivated mTOR. We propose susceptibility to ROS as the underlying cause for HSC’s general requirement for quiescence.     

Mitochondrial quality control by the ubiquitin-proteasome system

Mitochondria perform multiple functions critical to the maintenance of cellular homoeostasis and their dysfunction leads to disease. Several lines of evidence suggest the presence of a MAD (mitochondria-associated degradation) pathway that regulates mitochondrial protein quality control. Internal mitochondrial proteins may be retrotranslocated to the OMM (outer mitochondrial membrane), multiple E3 ubiquitin ligases reside at the OMM and inhibition of the proteasome causes accumulation of ubiquitinated proteins at the OMM. Reminiscent of ERAD [ER (endoplasmic reticulum)-associated degradation], Cdc48 (cell division cycle 42)/p97 is recruited to stressed mitochondria, extracts ubiquitinated proteins from the OMM and presents ubiquitinated proteins to the proteasome for degradation. Recent research has provided mechanistic insights into the interaction of the UPS (ubiquitin-proteasome system) with the OMM. In yeast, Vms1 [VCP (valosin-containing protein) (p97)/Cdc48-associated mitochondrial-stress-responsive 1] protein recruits Cdc48/p97 to the OMM. In mammalian systems, the E3 ubiquitin ligase parkin regulates the recruitment of Cdc48/p97 to mitochondria, subsequent mitochondrial protein degradation and mitochondrial autophagy. Disruption of the Vms1 or parkin systems results in the hyper-accumulation of ubiquitinated proteins at mitochondria and subsequent mitochondrial dysfunction. The emerging MAD pathway is important for the maintenance of cellular and therefore organismal viability.