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1.  During autophagy mitochondria elongate, are spared from degradation and sustain cell viability 
Nature cell biology  2011;13(5):589-598.
A plethora of cellular processes, including apoptosis, depend on regulated changes in mitochondrial shape and ultrastructure. Scarce is our understanding of the role of mitochondria and of their morphology during autophagy, a bulk degradation and recycling process of eukaryotic cells’ constituents. Here we show that mitochondrial morphology determines the cellular response to macroautophagy. When autophagy is triggered, mitochondria elongate in vitro and in vivo. Upon starvation cellular cAMP levels increase and protein kinase A (PKA) becomes activated. PKA in turn phosphorylates the pro-fission dynamin related protein 1 (DRP1) that is therefore retained in the cytoplasm, leading to unopposed mitochondrial fusion. Elongated mitochondria are spared from autophagic degradation, possess more cristae, increase dimerization and activity of ATP synthase, and maintain ATP production. When elongation is genetically or pharmacologically blocked, mitochondria conversely consume ATP, precipitating starvation-induced death. Thus, regulated changes in mitochondrial morphology determine the fate of the cell during autophagy.
PMCID: PMC3088644  PMID: 21478857
2.  Glucose metabolism determines resistance of cancer cells to bioenergetic crisis after cytochrome-c release 
How can cancer cells survive the consequences of cyt-c release? Huber et al provide a quantitative analysis of the protective role of enhanced glucose utilization in cancer cells and investigate the impact of cell-to-cell heterogeneity in mitochondrial bioenergetics.
How can cancer cells survive the consequences of cyt-c release? Huber et al provide a quantitative analysis of the protective role of enhanced glucose utilization in cancer cells and investigate the impact of cell-to-cell heterogeneity in mitochondrial bioenergetics.
We combine ordinary differential equations based computational modelling, single-cell microscopy and in biochemistry assays to provide the first integrated system study to portray the bioenergetic crisis in cell populations subsequent to cytochrome-c (cyt-c) release; a hallmark during chemotherapeutically induced cell death.We experimentally identified a cell-to-cell heterogeneity in the dynamics of the ATP synthase subsequent to cyt-c release, which the model explained by variations in (i) accessible cytochrome-c after release and (ii) the cell's glycolytic capacity.Our model predicted, and single-cell imaging confirmed, that high (increasing) glucose in media was able to sustain (repolarise) ΔΨm in HeLa cervical cancer and MCF-7 breast cancer cells, suggesting that they might recover from bioenergetic crisis upon elevation of glucose. However, no significant repolarisation was found in non-transformed human epithelial CRL-1807 cells. Therefore, this mechanism may provide cancer cells with a competitive advantage to evade cell death induced by anticancer drugs or other stress conditions when compared with non-transformed cells.
How can cells cope with a bioenergetic crisis? In particular, how can cancer cells survive the bioenergetic consequences of cyt-c release that are often induced by chemotherapeutic agents, and that lead to depolarisation of the mitochondrial membrane potential ΔΨm, result in loss of ionic homeostasis and induce cell death? Is there an inherent population heterogeneity that can lead to a non-synchronous response to above cell death stimuli, thereby aggravating treatment and contributing to clinical relapse? Do cancer cells have a competitive advantage to non-transformed cells in averting such a bioenergetic crisis after cyt-c release. We have investigated these questions in our study, which we regard as the first rigorous system study of single-cell bioenergetics subsequent to cyt-c release and one that bridges single-cell microscopy, in vitro analysis with ordinary differential equations (ODE) based modelling of bioenergetics pathways in the mitochondria and the cytosol.
Several laboratories have so far investigated cyt-c release experimentally (Slee et al, 1999; Atlante et al, 2000; Goldstein et al, 2000; Luetjens et al, 2001; Plas et al, 2001; Waterhouse et al, 2001; Ricci et al, 2003; Colell et al, 2007; Dussmann et al, 2003a, 2003b) and isolated mitochondria (Chinopoulos and Adam-Vizi, 2009; Kushnareva et al, 2002; Kushnareva et al, 2001). However, the cause and mechanistic of several key findings remain elusive and need a system level understanding of post-cyt-c release bioenergetic and its potential cross-talk to apoptosis signalling.
Ricci et al (2003) have identified that the cell death-inducing protease caspase-3, which get activated upon cyt-c release, can further impair mitochondrial function by cleaving and deactivating respiratory complexes I and II. We addressed the question of how such a mechanism could potentiate a bioenergetic crisis. To do so, we first assembled our ODE-based model by integrating approaches from metabolic modelling (Beard, 2005; Beard and Qian, 2007; Dash and Beard, 2008) and calibrated the model to literature data that describe bioenergetic state variables (mitochondrial membrane potential ΔΨm, mitochondrial transmembrane membrane ΔpH, respiration ratio between respiring and resting state mitochondria). By remodelling cyt-c release as observed experimentally and integrating it into our model as input, the single-cell model was able to correctly describe the kinetics of ΔΨm depolarisation and allowed its quantification. Moreover, it suggested that an additional complex I/II cleavage may further impair respiration and depolarise ΔΨm by approximately further 10%.
It was further reported that ATP synthase reversal, a change of direction in the ATP-producing enzyme that leads to pumping of protons from the mitochondrial matrix into the intramembrane space, can stabilise ΔΨm. By a remnant ΔΨm polarisation, cycling of Na+, Ca2+, K+, Cl− ions and protons across the mitochondrial and the plasma membranes is preserved, and ionic homeostasis can be maintained (Nicholls and Budd, 2000; Dussmann et al, 2003a; Chinopoulos and Adam-Vizi, 2009; Garedew et al, 2010). Our model confirmed that ATP synthase activity was reversed 10 min after onset of cyt-c release, predicted that ATP synthase reversal consumed ATP and that glycolysis was required and sufficient to provide the necessary ATP to sustain this reversal. Reverting back to our single-cell HeLa system, we confirmed the presence of ATP synthase reversal. However, reversal was only present in 20% of cells, 65% of cells showed no detectable reaction and even 15% maintained ATP synthase in forward direction.
To explain this cell-to-cell heterogeneity, we modelled that a cyt-c fraction remains accessible by respiratory complexes and for respiration subsequent to release, which we denoted as ‘respiration-accessible cyt-c'. Our model suggested that small variations in such levels can sufficiently explain the experimentally detected population heterogeneity in the direction and amount of ATP synthase proton flux (Figure 6AB). Variations in respiration-accessible cyt-c may arise from incomplete mitochondrial release. Such incomplete release has been associated with failure of cristae remodelling in the absence of the BH3-only family member BID or the intramitochondrial protein OPA1 (Frezza et al, 2006; Scorrano et al, 2002).
As the model identified glycolysis as necessary for sustaining ATP synthase reversal, we next investigated cells cultured in a medium that contained Na-pyruvate instead of glucose and which consequently were not able to perform glycolysis. We found that such cell populations had a significantly higher fraction of cells that maintained ATP synthase in forward mode consistent with our model predictions. The common influence of respiration-accessible cyt-c and the cell's ability to perform glycolysis is summarised in Figure 7A.
Because glycolysis was able to influence ATP synthase proton pumping, which can affect ΔΨm levels, we investigating the effect of higher glucose in single cells. Our model predicted that an increase in glucose utilisation that generates higher cytosolic ATP levels is able to stabilise and repolarise ΔΨm and after release. This mechanism is independent from ATP synthase direction. For cells that have ATP synthase in reverse mode, elevated ATP leads to increased proton efflux from the matrix, cell populations that maintain ATP synthase in forward mode achieve a similar result through a reduction of proton influx at increased ATP. In both cases, the proton gradient along the inner membrane, and therefore ΔΨm, is increased as a consequence of ATP elevation. The mechanism is depicted in Figure 7B.
We confirmed our model predictions that high glucose was able to stabilise (cells maintained in high-glucose media) and/or to repolarise (cells where glucose was added subsequent to release) ΔΨm (Figure 6). While a similar recovery was also present in MCF7 breast cancer cell lines, no significant effect of elevated glucose was found in non-transformed CRL-1807 cells. In conjunction with an impairment of caspase-dependent cell death observed in many human cancers, cancer cells may use this mechanism, and this mechanism may provide cancer cells with a competitive advantage to evade cell death induced by anticancer drugs or other stress conditions when compared with non-transformed cells.
Many anticancer drugs activate caspases via the mitochondrial apoptosis pathway. Activation of this pathway triggers a concomitant bioenergetic crisis caused by the release of cytochrome-c (cyt-c). Cancer cells are able to evade these processes by altering metabolic and caspase activation pathways. In this study, we provide the first integrated system study of mitochondrial bioenergetics and apoptosis signalling and examine the role of mitochondrial cyt-c release in these events. In accordance with single-cell experiments, our model showed that loss of cyt-c decreased mitochondrial respiration by 95% and depolarised mitochondrial membrane potential ΔΨm from −142 to −88 mV, with active caspase-3 potentiating this decrease. ATP synthase was reversed under such conditions, consuming ATP and stabilising ΔΨm. However, the direction and level of ATP synthase activity showed significant heterogeneity in individual cancer cells, which the model explained by variations in (i) accessible cyt-c after release and (ii) the cell's glycolytic capacity. Our results provide a quantitative and mechanistic explanation for the protective role of enhanced glucose utilisation for cancer cells to avert the otherwise lethal bioenergetic crisis associated with apoptosis initiation.
PMCID: PMC3094064  PMID: 21364572
apoptosis; bioenergetics; cancer; ODE; single-cell imaging
3.  Loss of Macroautophagy Promotes or Prevents Fibroblast Apoptosis Depending on the Death Stimulus*S◆ 
The Journal of biological chemistry  2007;283(8):4766-4777.
Macroautophagy has been implicated as a mechanism of cell death. However, the relationship between this degradative pathway and cell death is unclear as macroautophagy has been shown recently to protect against apoptosis. To better define the inter-play between these two critical cellular processes, we determined whether inhibition of macroautophagy could have both pro-apoptotic and anti-apoptotic effects in the same cell. Embryonic fibroblasts from mice with a knock-out of the essential macroautophagy gene atg5 were treated with activators of the extrinsic and intrinsic death pathways. Loss of macroautophagy sensitized these cells to caspase-dependent apoptosis from the death receptor ligands Fas and tumor necrosis factor-α (TNF-α). Atg5−/− mouse embryonic fibroblasts had increased activation of the mitochondrial death pathway in response to Fas/TNF-α in concert with decreased ATP levels. Fas/TNF-α treatment failed to up-regulate macroautophagy, and in fact, decreased activity at late time points. In contrast to their sensitization to Fas/TNF-α, Atg5−/− cells were resistant to death from menadione and UV light. In the absence of macroautophagy, an up-regulation of chaperone-mediated autophagy induced resistance to these stressors. These results demonstrate that inhibition of macroautophagy can promote or prevent apoptosis in the same cell and that the response is governed by the nature of the death stimulus and compensatory changes in other forms of autophagy. Experimental findings that an inhibition of macroautophagy blocks apoptosis do not prove that autophagy mediates cell death as this effect may result from the protective up-regulation of other autophagic pathways such as chaperone-mediated autophagy.
PMCID: PMC2754125  PMID: 18073215
4.  Mitophagy in neurodegeneration and aging 
Frontiers in Genetics  2012;3:297.
Macroautophagy is a cellular catabolic process that involves the sequestration of cytoplasmic constituents into double-membrane vesicles known as autophagosomes, which subsequently fuse with lysosomes, where they deliver their cargo for degradation. The main physiological role of autophagy is to recycle intracellular components, under conditions of nutrient deprivation, so as to supply cells with vital materials and energy. Selective autophagy also takes place in nutrient-rich conditions to rid the cell of damaged organelles or protein aggregates that would otherwise compromise cell viability. Mitophagy is a selective type of autophagy, whereby damaged or superfluous mitochondria are eliminated to maintain proper mitochondrial numbers and quality control. While mitophagy shares key regulatory factors with the general macroautophagy pathway, it also involves distinct steps, specific for mitochondrial elimination. Recent findings indicate that parkin and the phosphatase and tensin homolog-induced putative kinase protein 1 (PINK1), which have been implicated in the pathogenesis of neurodegenerative diseases such as Parkinson’s disease, also regulate mitophagy and function to maintain mitochondrial homeostasis. Here, we survey the molecular mechanisms that govern the process of mitophagy and discuss its involvement in the onset and progression of neurodegenerative diseases during aging.
PMCID: PMC3525948  PMID: 23267366
aging; autophagy; neuron; mitochondria; mitophagy; neurodegeneration; parkin; PINK1
5.  Macroautophagy regulates energy metabolism during effector T cell activation 
Macroautophagy is a highly conserved mechanism of lysosomal mediated protein degradation that plays a key role in maintaining cellular homeostasis by recycling amino acids, reducing the amount of damaged proteins, and regulating protein levels in response to extracellular signals. We have found that macroautophagy is induced following effector T cell activation. Engagement of the T cell receptor and CD28 results in enhanced LC3 processing, increased numbers of LC3-containing vesicles and increased LC3 flux, indicating active autophagosome formation and clearance. The autophagosomes formed in stimulated T cells actively fuse with lysosomes to degrade their cargo. Using a conditional knockout mouse model where Atg7, a critical gene for macroautophagy, is specifically deleted in T cells, we have found that macroautophagy-deficient effector T helper cells have defective IL-2 and INFγ production and reduced proliferation following stimulation, with no significant increase in apoptosis. We have found that ATP generation is decreased when autophagy is blocked, and defects in activation-induced cytokine production are restored when an exogenous energy source is added to macroautophagy-deficient T cells. Furthermore, we present evidence showing that the nature of the cargo inside autophagic vesicles found in resting T cells differs from the cargo of autophagosomes in activated T-cells, where mitochondria and other organelles are selectively excluded. These results suggest that macroautophagy is an actively regulated process in T cells that can be induced in response to T cell receptor engagement to accommodate the bioenergetic requirements of activated T cells.
PMCID: PMC3046774  PMID: 21059894
6.  Loss of Prohibitin Membrane Scaffolds Impairs Mitochondrial Architecture and Leads to Tau Hyperphosphorylation and Neurodegeneration 
PLoS Genetics  2012;8(11):e1003021.
Fusion and fission of mitochondria maintain the functional integrity of mitochondria and protect against neurodegeneration, but how mitochondrial dysfunctions trigger neuronal loss remains ill-defined. Prohibitins form large ring complexes in the inner membrane that are composed of PHB1 and PHB2 subunits and are thought to function as membrane scaffolds. In Caenorhabditis elegans, prohibitin genes affect aging by moderating fat metabolism and energy production. Knockdown experiments in mammalian cells link the function of prohibitins to membrane fusion, as they were found to stabilize the dynamin-like GTPase OPA1 (optic atrophy 1), which mediates mitochondrial inner membrane fusion and cristae morphogenesis. Mutations in OPA1 are associated with dominant optic atrophy characterized by the progressive loss of retinal ganglion cells, highlighting the importance of OPA1 function in neurons. Here, we show that neuron-specific inactivation of Phb2 in the mouse forebrain causes extensive neurodegeneration associated with behavioral impairments and cognitive deficiencies. We observe early onset tau hyperphosphorylation and filament formation in the hippocampus, demonstrating a direct link between mitochondrial defects and tau pathology. Loss of PHB2 impairs the stability of OPA1, affects mitochondrial ultrastructure, and induces the perinuclear clustering of mitochondria in hippocampal neurons. A destabilization of the mitochondrial genome and respiratory deficiencies manifest in aged neurons only, while the appearance of mitochondrial morphology defects correlates with tau hyperphosphorylation in the absence of PHB2. These results establish an essential role of prohibitin complexes for neuronal survival in vivo and demonstrate that OPA1 stability, mitochondrial fusion, and the maintenance of the mitochondrial genome in neurons depend on these scaffolding proteins. Moreover, our findings establish prohibitin-deficient mice as a novel genetic model for tau pathologies caused by a dysfunction of mitochondria and raise the possibility that tau pathologies are associated with other neurodegenerative disorders caused by deficiencies in mitochondrial dynamics.
Author Summary
Mitochondria are the major site of cellular ATP production and are essential for the survival of neurons. High ATP levels are required to sustain neuronal activities and axonal transport of macromolecules and organelles. The functional integrity of mitochondria depends on fusion and fission of their membranes, which maintain a dynamic mitochondrial network in cells. Interference with these processes causes neurodegenerative disorders that are characterized by axonal degeneration of distinct neurons. However, how an impaired fusion affects mitochondrial activities and neuronal survival remains poorly understood. Here, we have addressed this question by analyzing forebrain-specific knockout mice lacking prohibitins. Prohibitin complexes form membrane scaffolds in the inner membrane, which we now show are required for mitochondrial fusion, ultrastructure, and genome stability in neurons. Loss of prohibitins triggers extensive neurodegeneration associated with behavioral and cognitive deficiencies. Surprisingly, we observe hyperphosphorylation and filament formation of the microtubule-associated protein tau, reminiscent of a large group of neurodegenerative disorders termed tauopathies. Our findings, therefore, not only provide new insight into how defects in mitochondrial fusion affect neuronal survival, but also point to an intimate relationship of deficiencies in mitochondrial dynamics and tau pathologies.
PMCID: PMC3493444  PMID: 23144624
7.  Together we are stronger 
Autophagy  2011;7(12):1568-1569.
Starvation induces a protective process of self-cannibalization called autophagy that is thought to mediate nonselective degradation of cytoplasmic material. We recently reported that mitochondria escape autophagosomal degradation through extensive fusion into mitochondrial networks upon certain starvation conditions. The extent of mitochondrial elongation is dependent on the type of nutrient deprivation, with amino acid depletion having a particularly strong effect. Downregulation of the mitochondrial fission protein Drp1 was determined to be important in bringing about starvation-induced mitochondrial fusion. The formation of mitochondrial networks during nutrient depletion selectively blocked their autophagic degradation, presumably allowing cells to sustain efficient ATP production and thereby survive starvation.
PMCID: PMC3327623  PMID: 22024745
autophagy; Drp1; fission; fusion; mitochondria; PKA; starvation
8.  Francisella tularensis Harvests Nutrients Derived via ATG5-Independent Autophagy to Support Intracellular Growth 
PLoS Pathogens  2013;9(8):e1003562.
Francisella tularensis is a highly virulent intracellular pathogen that invades and replicates within numerous host cell types including macrophages, hepatocytes and pneumocytes. By 24 hours post invasion, F. tularensis replicates up to 1000-fold in the cytoplasm of infected cells. To achieve such rapid intracellular proliferation, F. tularensis must scavenge large quantities of essential carbon and energy sources from the host cell while evading anti-microbial immune responses. We found that macroautophagy, a eukaryotic cell process that primarily degrades host cell proteins and organelles as well as intracellular pathogens, was induced in F. tularensis infected cells. F. tularensis not only survived macroautophagy, but optimal intracellular bacterial growth was found to require macroautophagy. Intracellular growth upon macroautophagy inhibition was rescued by supplying excess nonessential amino acids or pyruvate, demonstrating that autophagy derived nutrients provide carbon and energy sources that support F. tularensis proliferation. Furthermore, F. tularensis did not require canonical, ATG5-dependent autophagy pathway induction but instead induced an ATG5-independent autophagy pathway. ATG5-independent autophagy induction caused the degradation of cellular constituents resulting in the release of nutrients that the bacteria harvested to support bacterial replication. Canonical macroautophagy limits the growth of several different bacterial species. However, our data demonstrate that ATG5-independent macroautophagy may be beneficial to some cytoplasmic bacteria by supplying nutrients to support bacterial growth.
Author Summary
Francisella tularensis is a highly virulent bacterial pathogen that infects hundreds of different animal species including humans. During infection, F. tularensis bacteria invade and rapidly multiply inside host cells. Within the host cell environment, basic nutrients that bacteria require for growth are in limited supply, and the majority of nutrients are tied up in complex molecules that are not readily available in forms that can be used by bacteria. In this study we asked and answered a very simple question; how does F. tularensis harvest sufficient carbon and energy sources from the host cell to support rapid intracellular growth? We found that F. tularensis induces a host recycling pathway in infected cells. Thus the host cell degrades nonessential proteins and releases amino acids. F. tularensis harvests the host-derived amino acids to generate energy and build its own more complex molecules. When we inhibited the host recycling pathway, growth of the intracellular bacteria was limited. Therefore, manipulation of host cell metabolism may be a means by which we can control the growth of intracellular bacterial pathogens during infection.
PMCID: PMC3744417  PMID: 23966861
9.  Optimal Dynamics for Quality Control in Spatially Distributed Mitochondrial Networks 
PLoS Computational Biology  2013;9(7):e1003108.
Recent imaging studies of mitochondrial dynamics have implicated a cycle of fusion, fission, and autophagy in the quality control of mitochondrial function by selectively increasing the membrane potential of some mitochondria at the expense of the turnover of others. This complex, dynamical system creates spatially distributed networks that are dependent on active transport along cytoskeletal networks and on protein import leading to biogenesis. To study the relative impacts of local interactions between neighboring mitochondria and their reorganization via transport, we have developed a spatiotemporal mathematical model encompassing all of these processes in which we focus on the dynamics of a health parameter meant to mimic the functional state of mitochondria. In agreement with previous models, we show that both autophagy and the generation of membrane potential asymmetry following a fusion/fission cycle are required for maintaining a healthy mitochondrial population. This health maintenance is affected by mitochondrial density and motility primarily through changes in the frequency of fusion events. Health is optimized when the selectivity thresholds for fusion and fission are matched, providing a mechanistic basis for the observed coupling of the two processes through the protein OPA1. We also demonstrate that the discreteness of the components exchanged during fusion is critical for quality control, and that the effects of limiting total amounts of autophagy and biogenesis have distinct consequences on health and population size, respectively. Taken together, our results show that several general principles emerge from the complexity of the quality control cycle that can be used to focus and interpret future experimental studies, and our modeling framework provides a road-map for deconstructing the functional importance of local interactions in communities of cells as well as organelles.
Author Summary
Mitochondria are the powerhouses of eukaryotic cells, oxidizing glucose to produce ATP. Most cells harbor tens to hundreds of mitochondria in a constant state of flux, in which they fuse with one another, undergo fission, import proteins to grow larger, and eventually are recycled by autophagy. These dynamic processes depend on the electrical potential that is maintained across the mitochondrial inner membrane and powers the production of both ATP and detrimental reactive oxygen species. How do mitochondria maintain high membrane potential in the face of damage due to reactive oxygen species? Here, we develop a model to study how the reorganization of mitochondrial networks in space and time due to fusion, fission, and the experimentally observed development of membrane potential asymmetry after fission affect overall mitochondrial health. We show that health, which is a proxy for the mitochondrial membrane potential, is dominated by how density and motility affect the frequency of fusion events, and that several simple rules for the system kinetics lead to optimal quality control. This model predicts general behaviors that can be applied to specific studies of mitochondrial dynamics in a wide variety of cell types, and provides a framework for deconstructing complex organellar organization and their function in human disease.
PMCID: PMC3708874  PMID: 23874166
10.  The Mitochondrial Inner Membrane Protein Mitofilin Controls Cristae MorphologyD⃞ 
Molecular Biology of the Cell  2005;16(3):1543-1554.
Mitochondria are complex organelles with a highly dynamic distribution and internal organization. Here, we demonstrate that mitofilin, a previously identified mitochondrial protein of unknown function, controls mitochondrial cristae morphology. Mitofilin is enriched in the narrow space between the inner boundary and the outer membranes, where it forms a homotypic interaction and assembles into a large multimeric protein complex. Down-regulation of mitofilin in HeLa cells by using specific small interfering RNA lead to decreased cellular proliferation and increased apoptosis, suggesting abnormal mitochondrial function. Although gross mitochondrial fission and fusion seemed normal, ultrastructural studies revealed disorganized mitochondrial inner membrane. Inner membranes failed to form tubular or vesicular cristae and showed as closely packed stacks of membrane sheets that fused intermittently, resulting in a complex maze of membranous network. Electron microscopic tomography estimated a substantial increase in inner:outer membrane ratio, whereas no cristae junctions were detected. In addition, mitochondria subsequently exhibited increased reactive oxygen species production and membrane potential. Although metabolic flux increased due to mitofilin deficiency, mitochondrial oxidative phosphorylation was not increased accordingly. We propose that mitofilin is a critical organizer of the mitochondrial cristae morphology and thus indispensable for normal mitochondrial function.
PMCID: PMC551514  PMID: 15647377
11.  Acidosis overrides oxygen deprivation to maintain mitochondrial function and cell survival 
Nature Communications  2014;5:3550.
Sustained cellular function and viability of high-energy demanding post-mitotic cells rely on the continuous supply of ATP. The utilization of mitochondrial oxidative phosphorylation for efficient ATP generation is a function of oxygen levels. As such, oxygen deprivation, in physiological or pathological settings, has profound effects on cell metabolism and survival. Here we show that mild extracellular acidosis, a physiological consequence of anaerobic metabolism, can reprogramme the mitochondrial metabolic pathway to preserve efficient ATP production regardless of oxygen levels. Acidosis initiates a rapid and reversible homeostatic programme that restructures mitochondria, by regulating mitochondrial dynamics and cristae architecture, to reconfigure mitochondrial efficiency, maintain mitochondrial function and cell survival. Preventing mitochondrial remodelling results in mitochondrial dysfunction, fragmentation and cell death. Our findings challenge the notion that oxygen availability is a key limiting factor in oxidative metabolism and brings forth the concept that mitochondrial morphology can dictate the bioenergetic status of post-mitotic cells.
In hypoxic conditions, cells depend on anaerobic respiration, which results in extracellular acidosis. Khacho et al. find that acidosis serves a protective function, enhancing mitochondrial respiratory capacity and sustaining ATP synthesis despite limited oxygen availability, by both promoting mitochondrial fusion and inhibiting fission.
PMCID: PMC3988820  PMID: 24686499
12.  Mitochondrial Turnover and Aging of Long-Lived Postmitotic Cells: The Mitochondrial–Lysosomal Axis Theory of Aging 
Antioxidants & Redox Signaling  2010;12(4):503-535.
It is now generally accepted that aging and eventual death of multicellular organisms is to a large extent related to macromolecular damage by mitochondrially produced reactive oxygen species, mostly affecting long-lived postmitotic cells, such as neurons and cardiac myocytes. These cells are rarely or not at all replaced during life and can be as old as the whole organism. The inherent inability of autophagy and other cellular-degradation mechanisms to remove damaged structures completely results in the progressive accumulation of garbage, including cytosolic protein aggregates, defective mitochondria, and lipofuscin, an intralysosomal indigestible material. In this review, we stress the importance of crosstalk between mitochondria and lysosomes in aging. The slow accumulation of lipofuscin within lysosomes seems to depress autophagy, resulting in reduced turnover of effective mitochondria. The latter not only are functionally deficient but also produce increased amounts of reactive oxygen species, prompting lipofuscinogenesis. Moreover, defective and enlarged mitochondria are poorly autophagocytosed and constitute a growing population of badly functioning organelles that do not fuse and exchange their contents with normal mitochondria. The progress of these changes seems to result in enhanced oxidative stress, decreased ATP production, and collapse of the cellular catabolic machinery, which eventually is incompatible with survival. Antioxid. Redox Signal. 12, 503–535.
ROS, Mitochondrial Damage, and Aging
Biomolecular damage under normal conditions
Imperfect turnover of damaged biologic structures
Major targets of ROS attack: mitochondria and lysosomes
Mitochondrial Fusion, Fission, and Biogenesis
The role of mitochondrial dynamics
Mitochondrial fusion
Mitochondrial fission
Mitochondrial biogenesis
Mitochondrial Proteolytic Systems
Mitochondrial Turnover by Autophagy
The main functions of the lysosomal compartment
Autophagic degradation of mitochondria (mitophagy)
Lipofuscin Formation and Its Influence on Autophagy
Influence of labile iron and ROS on lipofuscin formation
Consequences of the nondegradability of lipofuscin
Disease-related accumulation of intralysosomal and extralysosomal waste
Imperfect Mitochondrial Turnover and Postmitotic Cellular Aging
Age-related accumulation of defective mitochondria within postmitotic cells
Age-related decline in autophagy and Lon protease activity accelerates mitochondrial damage
Enlarged mitochondria are resistant to degradation and do not fuse with normal ones
Mechanisms of the age-related accumulation of mitochondria with homoplasmic mtDNA mutations
Decreased mitochondrial biogenesis in aged cells
Summary and Conclusions
PMCID: PMC2861545  PMID: 19650712
13.  Mechanism of Neuroprotective Mitochondrial Remodeling by PKA/AKAP1 
PLoS Biology  2011;9(4):e1000612.
The mitochondrial signaling complex PKA/AKAP1 protects neurons against mitochondrial fragmentation and cell death by phosphorylating and inactivating the mitochondrial fission enzyme Drp1.
Mitochondrial shape is determined by fission and fusion reactions catalyzed by large GTPases of the dynamin family, mutation of which can cause neurological dysfunction. While fission-inducing protein phosphatases have been identified, the identity of opposing kinase signaling complexes has remained elusive. We report here that in both neurons and non-neuronal cells, cAMP elevation and expression of an outer-mitochondrial membrane (OMM) targeted form of the protein kinase A (PKA) catalytic subunit reshapes mitochondria into an interconnected network. Conversely, OMM-targeting of the PKA inhibitor PKI promotes mitochondrial fragmentation upstream of neuronal death. RNAi and overexpression approaches identify mitochondria-localized A kinase anchoring protein 1 (AKAP1) as a neuroprotective and mitochondria-stabilizing factor in vitro and in vivo. According to epistasis studies with phosphorylation site-mutant dynamin-related protein 1 (Drp1), inhibition of the mitochondrial fission enzyme through a conserved PKA site is the principal mechanism by which cAMP and PKA/AKAP1 promote both mitochondrial elongation and neuronal survival. Phenocopied by a mutation that slows GTP hydrolysis, Drp1 phosphorylation inhibits the disassembly step of its catalytic cycle, accumulating large, slowly recycling Drp1 oligomers at the OMM. Unopposed fusion then promotes formation of a mitochondrial reticulum, which protects neurons from diverse insults.
Author Summary
Mitochondria, the cellular powerhouse, are highly dynamic organelles shaped by opposing fission and fusion events. Research over the past decade has identified many components of the mitochondrial fission/fusion machinery and led to the discovery that mutations in genes coding for these proteins can cause human neurological diseases. While it is well established that mitochondrial shape changes are intimately involved in cellular responses to environmental stressors, we know very little about the mechanisms by which cells dynamically adjust mitochondrial form and function. In this report, we show that the scaffold protein AKAP1 brings the cAMP-dependent protein kinase PKA to the outer mitochondrial membrane to protect neurons from injury. The PKA/AKAP1 complex functions by inhibiting Drp1, an enzyme that mechanically constricts and eventually severs mitochondria. Whereas active, dephosphorylated Drp1 rapidly cycles between cytosol and mitochondria, phosphorylated Drp1 builds up in inactive mitochondrial complexes, allowing mitochondria to fuse into a neuroprotective reticulum. Our results suggest that altering the balance of kinase and phosphatase activities at the outer mitochondrial membrane may provide the basis for novel neuroprotective therapies.
PMCID: PMC3079583  PMID: 21526220
14.  Acute focal brain damage alters mitochondrial dynamics and autophagy in axotomized neurons 
Cell Death & Disease  2014;5(11):e1545-.
Mitochondria are key organelles for the maintenance of life and death of the cell, and their morphology is controlled by continual and balanced fission and fusion dynamics. A balance between these events is mandatory for normal mitochondrial and neuronal function, and emerging evidence indicates that mitochondria undergo extensive fission at an early stage during programmed cell death in several neurodegenerative diseases. A pathway for selective degradation of damaged mitochondria by autophagy, known as mitophagy, has been described, and is of particular importance to sustain neuronal viability. In the present work, we analyzed the effect of autophagy stimulation on mitochondrial function and dynamics in a model of remote degeneration after focal cerebellar lesion. We provided evidence that lesion of a cerebellar hemisphere causes mitochondria depolarization in axotomized precerebellar neurons associated with PTEN-induced putative kinase 1 accumulation and Parkin translocation to mitochondria, block of mitochondrial fusion by Mfn1 degradation, increase of calcineurin activity and dynamin-related protein 1 translocation to mitochondria, and consequent mitochondrial fission. Here we suggest that the observed neuroprotective effect of rapamycin is the result of a dual role: (1) stimulation of autophagy leading to damaged mitochondria removal and (2) enhancement of mitochondria fission to allow their elimination by mitophagy. The involvement of mitochondrial dynamics and mitophagy in brain injury, especially in the context of remote degeneration after acute focal brain damage, has not yet been investigated, and these findings may offer new target for therapeutic intervention to improve functional outcomes following acute brain damage.
PMCID: PMC4260762  PMID: 25429622
15.  Mitochondrial autophagy in neural function, neurodegenerative disease, neuron cell death, and aging 
Neurobiology of disease  2010;43(1):46-51.
Macroautophagy is a cellular process by which cytosolic components and organelles are degraded in double-membrane bound structures upon fusion with lysosomes. A pathway for selective degradation of mitochondria by autophagy, known as mitophagy, has been described, and is of particular importance to neurons, because of the constant need for high levels of energy production in this cell type. Although much remains to be learned about mitophagy, it appears that the regulation of mitophagy shares key steps with the macroautophagy pathway, while exhibiting distinct regulatory steps specific for mitochondrial autophagic turnover. Mitophagy is emerging as an important pathway in neurodegenerative disease, and has been linked to the pathogenesis of Parkinson’s disease through the study of recessively inherited forms of this disorder, involving PINK1 and Parkin. Recent work indicates that PINK1 and Parkin together maintain mitochondrial quality control by regulating mitophagy. In the Purkinje cell degeneration (pcd) mouse, altered mitophagy may contribute to the dramatic neuron cell death observed in the cerebellum, suggesting that over-active mitophagy or insufficient mitophagy can both be deleterious. Finally, mitophagy has been linked to aging, as impaired macroautophagy over time promotes mitochondrial dysfunction associated with the aging process. Understanding the role of mitophagy in neural function, neurodegenerative disease, and aging represents an essential goal for future research in the autophagy field.
PMCID: PMC3096708  PMID: 20887789
16.  Mitochondria directly donate their membrane to form autophagosomes during a novel mechanism of parkin-associated mitophagy 
Cell & Bioscience  2014;4:16.
Autophagy (macroautophagy), a cellular process of “self-eating”, segregates damaged/aged organelles into vesicles, fuses with lysosomes, and enables recycling of the digested materials. The precise origin(s) of the autophagosome membrane is unclear and remains a critical but unanswered question. Endoplasmic reticulum, mitochondria, Golgi complex, and the plasma membrane have been proposed as the source of autophagosomal membranes.
Using electron microscopy, immunogold labeling techniques, confocal microscopy, and flow cytometry we show that mitochondria can directly donate their membrane material to form autophagosomes. We expand upon earlier studies to show that mitochondria donate their membranes to form autophagosomes during basal and drug-induced autophagy. Moreover, electron microscopy and immunogold labeling studies show the first physical evidence of mitochondria forming continuous structures with LC3-labeled autophagosomes. The mitochondria forming these structures also stain positive for parkin, indicating that these mitochondrial-formed autophagosomes represent a novel mechanism of parkin-associated mitophagy.
With the on-going debate regarding autophagosomal membrane origin, this report demonstrates that mitochondria can donate membrane materials to form autophagosomes. These structures may also represent a novel form of mitophagy where the mitochondria contribute to the formation of autophagosomes. This novel form of parkin-associated mitophagy may be a more efficient bio-energetic process compared with de novo biosynthesis of a new membrane, particularly if the membrane is obtained, at least partly, from the organelle being targeted for later degradation in the mature autolysosome.
PMCID: PMC3977894  PMID: 24669863
Breast cancer; Mitochondria; Autophagy; Mitophagy; Parkin; Antiestrogen resistance; Fulvestrant; Imatinib; Estrogen receptor-α
17.  Bcl-xL regulates mitochondrial energetics by stabilizing the inner membrane potential 
The Journal of Cell Biology  2011;195(2):263-276.
To promote cell survival, the antiapoptotic factor Bcl-xL both inhibits Bax-induced mitochondrial outer membrane permeabilization and stabilizes mitochondrial inner membrane ion flux and thus overall mitochondrial energetic capacity.
Mammalian Bcl-xL protein localizes to the outer mitochondrial membrane, where it inhibits apoptosis by binding Bax and inhibiting Bax-induced outer membrane permeabilization. Contrary to expectation, we found by electron microscopy and biochemical approaches that endogenous Bcl-xL also localized to inner mitochondrial cristae. Two-photon microscopy of cultured neurons revealed large fluctuations in inner mitochondrial membrane potential when Bcl-xL was genetically deleted or pharmacologically inhibited, indicating increased total ion flux into and out of mitochondria. Computational, biochemical, and genetic evidence indicated that Bcl-xL reduces futile ion flux across the inner mitochondrial membrane to prevent a wasteful drain on cellular resources, thereby preventing an energetic crisis during stress. Given that F1FO–ATP synthase directly affects mitochondrial membrane potential and having identified the mitochondrial ATP synthase β subunit in a screen for Bcl-xL–binding partners, we tested and found that Bcl-xL failed to protect β subunit–deficient yeast. Thus, by bolstering mitochondrial energetic capacity, Bcl-xL may contribute importantly to cell survival independently of other Bcl-2 family proteins.
PMCID: PMC3198165  PMID: 21987637
18.  Ubiquilin functions in autophagy and is degraded by chaperone-mediated autophagy 
Human Molecular Genetics  2010;19(16):3219-3232.
Autophagy is the process by which organelles and portions of the cytoplasm are degraded in lysosomes. Several different forms of autophagy are known that are distinguishable chiefly by the mode in which cargo is delivered to the lysosome for degradation. Ubiquilin was recently reported to regulate macroautophagy, the form of autophagy in which cytosolic cargo is packaged in a double-membrane structure or autophagosome that fuses with lysosomes for degradation. We confirm here using different morphological and biochemical procedures that ubiquilin is present in autophagosomes in HeLa cells and in brain and liver tissue of mouse. Coimmunoprecipitation studies indicated that ubiquilin binds the autophagosome marker LC3 in a complex and that reduction of ubiquilin expression reduces autophagosome formation, which correlates with a reduction in maturation of LC3-I to the LC3-II form of the protein. We found that ubiquilin is degraded during both macroautophagy and during chaperone-mediated autophagy (CMA), the latter of which involves the active transport of proteins into lysosomes. We discuss the implication of this degradation in mediating cross-talk between macroautophagy and CMA. Finally, we demonstrate that ubiquilin protects cells against starvation-induced cell death propagated by overexpression of mutant Alzheimer's disease PS2N141I protein and green fluorescent protein (GFP)-huntingtin exon-1 fusion protein containing 74 polyglutamines.
PMCID: PMC2908472  PMID: 20529957
19.  The inner membrane protein Mdm33 controls mitochondrial morphology in yeast 
The Journal of Cell Biology  2003;160(4):553-564.
Mitochondrial distribution and morphology depend on MDM33, a Saccharomyces cerevisiae gene encoding a novel protein of the mitochondrial inner membrane. Cells lacking Mdm33 contain ring-shaped, mostly interconnected mitochondria, which are able to form large hollow spheres. On the ultrastructural level, these aberrant organelles display extremely elongated stretches of outer and inner membranes enclosing a very narrow matrix space. Dilated parts of Δmdm33 mitochondria contain well-developed cristae. Overexpression of Mdm33 leads to growth arrest, aggregation of mitochondria, and generation of aberrant inner membrane structures, including septa, inner membrane fragments, and loss of inner membrane cristae. The MDM33 gene is required for the formation of net-like mitochondria in mutants lacking components of the outer membrane fission machinery, and mitochondrial fusion is required for the formation of extended ring-like mitochondria in cells lacking the MDM33 gene. The Mdm33 protein assembles into an oligomeric complex in the inner membrane where it performs homotypic protein–protein interactions. Our results indicate that Mdm33 plays a distinct role in the mitochondrial inner membrane to control mitochondrial morphology. We propose that Mdm33 is involved in fission of the mitochondrial inner membrane.
PMCID: PMC2173741  PMID: 12591915
membrane fission; mitochondria; mitochondrial dynamics; organelle morphology; Saccharomyces cerevisiae
20.  Defects in Mitochondrial Fission Protein Dynamin-Related Protein 1 Are Linked to Apoptotic Resistance and Autophagy in a Lung Cancer Model 
PLoS ONE  2012;7(9):e45319.
Evasion of apoptosis is implicated in almost all aspects of cancer progression, as well as treatment resistance. In this study, resistance to apoptosis was identified in tumorigenic lung epithelial (A549) cells as a consequence of defects in mitochondrial and autophagic function. Mitochondrial function is determined in part by mitochondrial morphology, a process regulated by mitochondrial dynamics whereby the joining of two mitochondria, fusion, inhibits apoptosis while fission, the division of a mitochondrion, initiates apoptosis. Mitochondrial morphology of A549 cells displayed an elongated phenotype–mimicking cells deficient in mitochondrial fission protein, Dynamin-related protein 1 (Drp1). A549 cells had impaired Drp1 mitochondrial recruitment and decreased Drp1-dependent fission. Cytochrome c release and caspase-3 and PARP cleavage were impaired both basally and with apoptotic stimuli in A549 cells. Increased mitochondrial mass was observed in A549 cells, suggesting defects in mitophagy (mitochondrial selective autophagy). A549 cells had decreased LC3-II lipidation and lysosomal inhibition suggesting defects in autophagy occur upstream of lysosomal degradation. Immunostaining indicated mitochondrial localized LC3 punctae in A549 cells increased after mitochondrial uncoupling or with a combination of mitochondrial depolarization and ectopic Drp1 expression. Increased inhibition of apoptosis in A549 cells is correlated with impeded mitochondrial fission and mitophagy. We suggest mitochondrial fission defects contribute to apoptotic resistance in A549 cells.
PMCID: PMC3447926  PMID: 23028930
21.  Mitochondrial autophagy as a compensatory response to PINK1 deficiency 
Autophagy  2009;5(8):1213-1214.
Macroautophagy (hereafter, autophagy) plays a critical role in maintaining cellular homeostasis by degrading protein aggregates and dysfunctional/damaged organelles. We recently reported that silencing the recessive familial Parkinson disease gene encoding PTEN-induced kinase 1 (PINK1) leads to neuronal cell death accompanied by mitochondrial dysfunction and Drp1-dependent fragmentation. In this model, mitochondrial fission and Beclin 1-dependent autophagy play protective roles, cooperating to sequester and eliminate damaged mitochondria. We discuss the role of superoxide and other reactive oxygen species upstream of mitochondrial depolarization, fission and autophagy in PINK1 knockdown lines. PINK1 deficiency appears to trigger several compensatory responses that together facilitate clearance of depolarized mitochondria, through a mechanism that is further enhanced by increased expression of parkin. These data offer additional insights that broaden the spectrum of potential interactions between PINK1 and parkin with respect to the regulation of mitochondrial homeostasis and mitophagy.
PMCID: PMC2841445  PMID: 19786829
mitochondria; PINK1; autophagy; cell death; parkinson disease; parkin; beclin 1
22.  Rotenone Inhibits Autophagic Flux Prior to Inducing Cell Death 
ACS Chemical Neuroscience  2012;3(12):1063-1072.
Rotenone, which selectively inhibits mitochondrial complex I, induces oxidative stress, α-synuclein accumulation, and dopaminergic neuron death, principal pathological features of Parkinson's disease. The autophagy–lysosome pathway degrades damaged proteins and organelles for the intracellular maintenance of nutrient and energy balance. While it is known that rotenone causes autophagic vacuole accumulation, the mechanism by which this effect occurs has not been thoroughly investigated. Treatment of differentiated SH-SY5Y cells with rotenone (10 μM) induced the accumulation of autophagic vacuoles at 6 h and 24 h as indicated by Western blot analysis for microtubule associated protein-light chain 3-II (MAP-LC3-II). Assessment of autophagic flux at these time points indicated that autophagic vacuole accumulation resulted from a decrease in their effective lysosomal degradation, which was substantiated by increased levels of autophagy substrates p62 and α-synuclein. Inhibition of lysosomal degradation may be explained by the observed decrease in cellular ATP levels, which in turn may have caused the observed concomitant increase in acidic vesicle pH. The early (6 h) effects of rotenone on cellular energetics and autophagy–lysosome pathway function preceded the induction of cell death and apoptosis. These findings indicate that the classical mitochondrial toxin rotenone has a pronounced effect on macroautophagy completion that may contribute to its neurotoxic potential.
PMCID: PMC3526971  PMID: 23259041
Rotenone; autophagy; lysosome; cell death; Parkinson's disease; SH-SY5Y
23.  ChChd3, an Inner Mitochondrial Membrane Protein, Is Essential for Maintaining Crista Integrity and Mitochondrial Function* 
The Journal of Biological Chemistry  2010;286(4):2918-2932.
The mitochondrial inner membrane (IM) serves as the site for ATP production by hosting the oxidative phosphorylation complex machinery most notably on the crista membranes. Disruption of the crista structure has been implicated in a variety of cardiovascular and neurodegenerative diseases. Here, we characterize ChChd3, a previously identified PKA substrate of unknown function (Schauble, S., King, C. C., Darshi, M., Koller, A., Shah, K., and Taylor, S. S. (2007) J. Biol. Chem. 282, 14952–14959), and show that it is essential for maintaining crista integrity and mitochondrial function. In the mitochondria, ChChd3 is a peripheral protein of the IM facing the intermembrane space. RNAi knockdown of ChChd3 in HeLa cells resulted in fragmented mitochondria, reduced OPA1 protein levels and impaired fusion, and clustering of the mitochondria around the nucleus along with reduced growth rate. Both the oxygen consumption and glycolytic rates were severely restricted. Ultrastructural analysis of these cells revealed aberrant mitochondrial IM structures with fragmented and tubular cristae or loss of cristae, and reduced crista membrane. Additionally, the crista junction opening diameter was reduced to 50% suggesting remodeling of cristae in the absence of ChChd3. Analysis of the ChChd3-binding proteins revealed that ChChd3 interacts with the IM proteins mitofilin and OPA1, which regulate crista morphology, and the outer membrane protein Sam50, which regulates import and assembly of β-barrel proteins on the outer membrane. Knockdown of ChChd3 led to almost complete loss of both mitofilin and Sam50 proteins and alterations in several mitochondrial proteins, suggesting that ChChd3 is a scaffolding protein that stabilizes protein complexes involved in maintaining crista architecture and protein import and is thus essential for maintaining mitochondrial structure and function.
PMCID: PMC3024787  PMID: 21081504
Bioenergetics; Confocal Microscopy; Electron Microscopy (EM); Mitochondria; Protein-Protein Interactions; ChChd3; Crista Junctions; Crista Morphology; Mitochondrial Dynamics; Protein Import
24.  Modulation of mitochondrial function and morphology by interaction of Omi/HtrA2 with the mitochondrial fusion factor OPA1 
Experimental Cell Research  2010;316(7):1213-1224.
Loss of Omi/HtrA2 function leads to nerve cell loss in mouse models and has been linked to neurodegeneration in Parkinson's and Huntington's disease. Omi/HtrA2 is a serine protease released as a pro-apoptotic factor from the mitochondrial intermembrane space into the cytosol. Under physiological conditions, Omi/HtrA2 is thought to be involved in protection against cellular stress, but the cytological and molecular mechanisms are not clear. Omi/HtrA2 deficiency caused an accumulation of reactive oxygen species and reduced mitochondrial membrane potential. In Omi/HtrA2 knockout mouse embryonic fibroblasts, as well as in Omi/HtrA2 silenced human HeLa cells and Drosophila S2R+ cells, we found elongated mitochondria by live cell imaging. Electron microscopy confirmed the mitochondrial morphology alterations and showed abnormal cristae structure. Examining the levels of proteins involved in mitochondrial fusion, we found a selective up-regulation of more soluble OPA1 protein. Complementation of knockout cells with wild-type Omi/HtrA2 but not with the protease mutant [S306A]Omi/HtrA2 reversed the mitochondrial elongation phenotype and OPA1 alterations. Finally, co-immunoprecipitation showed direct interaction of Omi/HtrA2 with endogenous OPA1. Thus, we show for the first time a direct effect of loss of Omi/HtrA2 on mitochondrial morphology and demonstrate a novel role of this mitochondrial serine protease in the modulation of OPA1. Our results underscore a critical role of impaired mitochondrial dynamics in neurodegenerative disorders.
PMCID: PMC3063334  PMID: 20064504
ANT, adenine nucleotide translocator; Drp1, dynamin-related protein 1; Fis1, mitochondrial fission 1 protein; Hsp90, heat shock protein 90; HtrA2, high temperature requirement protein A2; KO, knockout; MEF, mouse embryonic fibroblast; Mfn2, mitofusin 2; MMP, mitochondrial membrane potential; PBS, phosphate-buffered saline; PD, Parkinson's disease; ROS, reactive oxygen species; SD, standard deviation; SEM, standard error of the mean; VDAC1, voltage dependent anion channel 1; WT, wild-type; Omi; HtrA2; Mitochondria; Fusion; OPA1; Parkinson's disease
Hepatology (Baltimore, Md.)  2010;52(1):266-277.
The function of the lysosomal degradative pathway of autophagy in cellular injury is unclear as findings in nonhepatic cells have implicated autophagy as both a mediator of cell death and as a survival response. Autophagic function is impaired in steatotic and aged hepatocytes, suggesting that in these settings hepatocellular injury may be altered by the decrease in autophagy. To delineate the specific function of autophagy in the hepatocyte injury response, the effects of menadione-induced oxidative stress were examined in the RALA255-10G rat hepatocyte line when macroautophagy was inhibited by an shRNA-mediated knockdown of the autophagy gene atg5. Inhibition of macroautophagy sensitized cells to apoptotic and necrotic death from normally nontoxic concentrations of menadione. Inhibition of macroautophagy led to overactivation of the c-Jun N-terminal kinase (JNK)/c-Jun signaling pathway that induced cell death. Death occurred from activation of the mitochondrial death pathway with cellular ATP depletion, mitochondrial cytochrome c release and caspase activation. Sensitization to death from menadione occurred despite up regulation of other forms of autophagy in compensation for the loss of macroautophagy. Chaperone-mediated autophagy (CMA) also mediated resistance to menadione as CMA inhibition sensitized cells to death from menadione through a mechanism different from that of a loss of macroautophagy as death occurred in the absence of JNK/c-Jun overactivation or ATP depletion.
Hepatocyte resistance to injury from menadione-induced oxidative stress is mediated by distinct functions of both macroautophagy and CMA, indicating that impaired function of either form of autophagy may promote oxidant-induced liver injury.
PMCID: PMC2924621  PMID: 20578144
apoptosis; necrosis; menadione; mitochondria; c-Jun N-terminal kinase

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