Abnormal mitochondrial autophagy in nephropathic cystinosis

Cystinosis, which is characterized by lysosomal accumulation of cystine in many tissues, was the first known storage disorder caused by defective metabolite export from the lysosome. The molecular and cellular mechanisms underlying nephropathic cystinosis, the most severe form, which exhibits generalized proximal tubular dysfunction and progressive renal failure, remain largely unknown. We used renal proximal tubular epithelial (RPTE) cells and fibroblasts from patients with three clinical variants of cystinosis: nephropathic, intermediate and ocular to explore the specific injury mechanism in nephropathic cystinosis. We demonstrate enhanced autophagy of mitochondria, increase in apoptosis and mitochondrial dysfunction in the nephropathic cystinosis phenotype. Furthermore, specific inhibition of autophagy results in significant attenuation of cell death in nephropathic cystinosis. This study provides ultrastructural and functional evidence of abnormal mitochondrial autophagy in nephropathic cystinosis, which may contribute to renal Fanconi syndrome and progressive renal injury.Key words: cystinosis, autophagy, mitochondria, kidney, lysosome, apoptosis, cell death, mitophagyCystinosis is an autosomal recessive metabolic disorder caused by mutations in the CTNS gene, which encodes a 7-transmembrane domain protein, cystinosin, a lysosomal cystine transporter. Cystinosis belongs to the family of lysosomal storage disorders (LSDs) characterized by the tissue accumulation of cystine crystals leading to multiple organ dysfunction. The three types of cystinosis, i.e., nephropathic (classic renal and systemic disease), intermediate (a late-onset variant of nephropathic cystinosis) and non-nephropathic (clinically affecting only the cornea) are allelic disorders caused by CTNS mutations. Children affected with nephropathic cystinosis present with the Fanconi syndrome and usually develop progressive renal failure within the first decade of life. The mechanism linking lysosomal cystine storage to pathological manifestations, in particular to the prominent proximal tubular defect and renal injury, remains unclear. Renal injury in nephropathic cystinosis may not simply be caused just by cystine accumulation, as disruption of the ctns gene in mice induces cystine storage in many tissues but does not result in signs of tubulopathy or renal failure; renal injury is not seen in other human forms of cystinosis and progressive renal injury occurs despite cystine depletion therapy.The purpose of our study was to investigate the specific mechanism leading to tubulopathy and end stage renal injury in nephropathic cystinosis. We used primary fibroblast and renal proximal tubular epithelial (RPTE) cells derived from patients with three clinical phenotypes of cystinosis. Our data show an abnormal increase in macroautophagy (hereafter referred to as autophagy), specific to the nephropathic variant of cystinosis. We also demonstrate that specific inhibition of autophagy rescues cell death in nephropathic cystinotic RPTE cells. Our results indicate that mitochondrial autophagy may be a critical mechanism contributing to renal Fanconi syndrome and progressive renal injury in nephropathic cystinosis.Abnormal autophagy was also recently observed in other types of lysosomal storage diseases (LSD). However, our study provides the first evidence supporting the extensive involvement of autophagy in nephropathic cystinosis pathogenesis. Abundant vacuolization and abnormal mitochondria are detected by electron microscopy (EM) in nephropathic cystinotic cells. Additionally, elevated levels of LC3-II and Beclin 1 are also observed in nephropathic cystinotic RPTE cells, indicating a role of Beclin 1-mediated autophagy in cystinosis. These results altogether establish an abnormal increase in autophagy in nephropathic cystinotic cells.Renal biopsies from patients with nephropathic cystinosis can reveal abnormally large mitochondria, but the relevance of this finding and other ultrastructural abnormalities is unclear. Our study further demonstrates a significant decrease in mitochondrial ATP generation with an increase in reactive oxygen species (ROS) in cystinotic cells. To further dissect the association of abnormal mitochondria with increased autophagy in cystinosis, we carefully examined the electron micrographs at higher magnifications. We discovered various stages of degradation of mitochondria by autophagy (hereafter referred to as mitophagy). To further validate mitophagy in cystinosis, we used an immunofluorescence (IF) approach to capture colocalization images of LC3, LAMP-2 (lysosomal marker) and ATP5H (mitochondrial marker). Intriguingly, an increase in LAMP-2 perinuclear staining is detected by IF assay in cystinotic cells. This observation may also denote enhanced active autophagy as LAMP-2 is involved in lysosomal biogenesis and/or the fusion between autophagosomes and lysosomes. Alternatively, LAMP-2 accumulation could be a manifestation of retarded autophagic flux in cystinotic cells. A decreased ability of lysosomes to fuse with autophagosomes has been reported in various LSDs. However, the colocalization of LC3 and LAMP-2 in nephropathic cystinotic RPTE cells argues against this possibility. Nevertheless, the possibility of autophagic flux blockade after autophagosome-lysosome fusion leading to detrimental effects is yet to be investigated. Interestingly, previously published EM reports of the renal biopsies of patients with nephropathic cystinosis show only the nucleus and a thin rim of cytoplasm as remnants in a proximal tubular cell, while mitochondria and lysosomes completely disappear.Conventionally, autophagy has been suggested as a cytoprotective mechanism to ensure cell survival during starvation. In contrast, several forms of cell death have been associated with the appearance of autophagic vesicles. To gain insight into the role of autophagy as regards to cell death or cell survival in nephropathic cystinosis, we used 3-methyladenine (3MA), a specific inhibitor of autophagy and assayed cell viability and apoptosis in cystinotic cells. Increased apoptosis has been previously reported in cultured cystinotic fibroblasts and RPTE cells. Treatment with 3MA in cystinotic cells significantly rescues cell death, thus suggesting a synergistic role of apoptosis and autophagy in cystinosis.In conclusion, as illustrated in Figure 1, we speculate that there is a multifaceted impact of autophagy in nephropathic cystinosis as follows: (1) the mechanism linking autophagy to lysosomal cystine or apoptosis in cystinotic cells could potentially be related to lysosomal membrane permeabilization (LMP), proposed as an early step in apoptosis in cystinosis. We hypothesize that abnormal induction of autophagy besides providing more cargo to be digested in the lysosomes, leads to increased fusion of autophagosomes with cystine-laden lysosomes, rendering them more sensitive to membrane destabilization, and thus making them readily enter the apoptotic pathway; (2) the second most important question is the link between abnormal mitochondria and mitophagy in cystinosis. A decreased level of cytosolic glutathione in cystinotic cells is one of the known factors responsible for generating damaged mitochondria. Our data also indicate an impairment of complex I activity, an increase in ROS and a decrease in mitochondrial ATP generation in cystinotic cells. We hypothesize that the abnormal induction of autophagy leads to depletion of mitochondria, forcing cells to enter the ‘starvation mode,’ thereby leading to an uncontrolled autophagy and cell death; (3) the third key question yet to be answered is the link between autophagy and renal injury in nephropathic cystinosis. Skeletal muscles and neuronal tissues are the primary organs where autophagy is physiologically enhanced. Recently, it has been shown that mouse kidneys exert a high level of autophagy under basal conditions, influencing the susceptibility to glomerular disease and renal failure. Thus, we postulate an organ- and tissue-specific effect of abnormally induced autophagy in nephropathic cystinosis, causing severe injury to kidneys leading to loss of renal function, ultimately culminating in end-stage renal disease.Open in a separate windowFigure 1A schematic view of the interplay between autophagy, abnormal mitochondria and cell death in cystinosis. Abnormal induction of autophagy, typically mitophagy, forces cells into a starvation mode leading to cell death; and renders cystine-laden lysosomes sensitive to lysosomal membrane permeabilization (LMP) making it readily enter the apoptosis pathway. A potential block in autophagic flux, after autophagosome-lysosome fusion, remains to be elucidated. Preferential severe kidney damage in nephropathic cystinosis may be due to the tissue- and organ-specific injury effect of autophagy.The recent progress in autophagy research has increased the need for additional studies so that we can fully understand the underlying pathological mechanisms and the significance of the lysosomal cell death axis in lysosomal storage disorders.     

Lysosomal enzymes promote mitochondrial oxidant production, cytochrome c release and apoptosis.

Exposure of mammalian cells to oxidant stress causes early (iron catalysed) lysosomal rupture followed by apoptosis or necrosis. Enhanced intracellular production of reactive oxygen species (ROS), presumably of mitochondrial origin, is also observed when cells are exposed to nonoxidant pro-apoptotic agonists of cell death. We hypothesized that ROS generation in this latter case might promote the apoptotic cascade and could arise from effects of released lysosomal materials on mitochondria. Indeed, in intact cells (J774 macrophages, HeLa cells and AG1518 fibroblasts) the lysosomotropic detergent O-methyl-serine dodecylamide hydrochloride (MSDH) causes lysosomal rupture, enhanced intracellular ROS production, and apoptosis. Furthermore, in mixtures of rat liver lysosomes and mitochondria, selective rupture of lysosomes by MSDH promotes mitochondrial ROS production and cytochrome c release, whereas MSDH has no direct effect on ROS generation by purifed mitochondria. Intracellular lysosomal rupture is associated with the release of (among other constituents) cathepsins and activation of phospholipase A2 (PLA2). We find that addition of purified cathepsins B or D, or of PLA2, causes substantial increases in ROS generation by purified mitochondria. Furthermore, PLA2 - but not cathepsins B or D - causes rupture of semipurified lysosomes, suggesting an amplification mechanism. Thus, initiation of the apoptotic cascade by nonoxidant agonists may involve early release of lysosomal constituents (such as cathepsins B and D) and activation of PLA2, leading to enhanced mitochondrial oxidant production, further lysosomal rupture and, finally, mitochondrial cytochrome c release. Nonoxidant agonists of apoptosis may, thus, act through oxidant mechanisms.     

Autophagy delays apoptotic death in breast cancer cells following DNA damage

Early signaling in camptothecin-treated MCF-7 cells followed an intrinsic pathway, but death was delayed and late events exhibited few hallmarks of apoptosis. BH3-only proteins, such as Noxa, Puma and BimEL, were activated and localized to mitochondrial sites within 24 h following drug exposure. However, caspase activity was low and death was unaffected by caspase inhibition. Transmission electron micrographs showed the presence of large vacuoles in drug-treated cells. An autophagic survival response has been attributed to MCF-7 cells following nutrient starvation or exposure to tamoxifen. Here, we show that autophagy also plays an important role in the delayed DNA damage response. Confocal microscopy revealed colocalization of mitochondria with large autophagic vacuoles and inhibitors of autophagy increased mitochondrial depolarization and caspase-9 activity, and accelerated cell death. Furthermore, downregulation of autophagy proteins, Beclin 1 and Atg7, unmasked a caspase-dependent, apoptotic response to DNA damage. We propose that a post-mitochondrial caspase cascade is delayed as a result of early disposal of damaged mitochondria within autophagosomes. Our data also suggest that the use of autophagy as a means of delaying apoptosis or prolonging survival may be characteristic of noninvasive breast tumor cells. These studies underscore a potential role for autophagy inhibitors in combination with conventional chemotherapeutic drugs in early breast cancer therapy.     

Anti-cancer agent siramesine is a lysosomotropic detergent that induces cytoprotective autophagosome accumulation

A σ-2 receptor ligand siramesine induces lysosomal leakage and cathepsin-dependent death of cancer cells in vitro and displays potent anti-cancer activity in vivo. The mechanism by which siramesine destabilizes lysosomes is, however, unknown. Here, we show that siramesine induces a rapid rise in the lysosomal pH that is followed by lysosomal leakage and dysfunction. The rapid accumulation of siramesine into cancer cell lysosomes, its ability to destabilize isolated lysosomes, and its chemical structure as an amphiphilic amine indicate that it is a lysosomotropic detergent. Notably, siramesine triggers also a substantial Atg6- and Atg7-dependent accumulation of autophagosomes that is associated with a rapid and sustained inhibition of mammalian target of rapamycin complex 1 (mTORC1; an inhibitor of autophagy). Siramesine fails, however, to increase the degradation rate of long-lived proteins. Thus, the massive accumulation of autophagosomes is likely to be due to a combined effect of activation of autophagy signaling and decreased autophagosome turnover. Importantly, pharmacological and RNA interference-based inhibition of autophagosome formation further sensitizes cancer cells to siramesine-induced cytotoxicity. These data identify siramesine as a lysosomotropic detergent that triggers cell death via a direct destabilization of lysosomes and cytoprotection by inducing the accumulation of autophagosomes. Threrefore, the combination of siramesine with inhibitors of autophagosome formation appears as a promising approach for future cancer therapy.     

Anti-cancer agent siramesine is a lysosomotropic detergent that induces cytoprotective autophagosome accumulation

A sigma-2 receptor ligand siramesine induces lysosomal leakage and cathepsin-dependent death of cancer cells in vitro and displays potent anti-cancer activity in vivo. The mechanism by which siramesine destabilizes lysosomes is, however, unknown. Here, we show that siramesine induces a rapid rise in the lysosomal pH that is followed by lysosomal leakage and dysfunction. The rapid accumulation of siramesine into cancer cell lysosomes, its ability to destabilize isolated lysosomes, and its chemical structure as an amphiphilic amine indicate that it is a lysosomotropic detergent. Notably, siramesine triggers also a substantial Atg6- and Atg7-dependent accumulation of autophagosomes that is associated with a rapid and sustained inhibition of mammalian target of rapamycin complex 1 (mTORC1; an inhibitor of autophagy). Siramesine fails, however, to increase the degradation rate of long-lived proteins. Thus, the massive accumulation of autophagosomes is likely to be due to a combined effect of activation of autophagy signaling and decreased autophagosome turnover. Importantly, pharmacological and RNA interference-based inhibition of autophagosome formation further sensitizes cancer cells to siramesine-induced cytotoxicity. These data identify siramesine as a lysosomotropic detergent that triggers cell death via a direct destabilization of lysosomes and cytoprotection by inducing the accumulation of autophagosomes. Threrefore, the combination of siramesine with inhibitors of autophagosome formation appears as a promising approach for future cancer therapy.     

Is mitochondrial generation of reactive oxygen species a trigger for autophagy?

Autophagy is a conserved lysosomal degradation pathway that has been extensively studied in recent years. However, the mechanism of autophagy induction is still not clear. Mitochondria are important regulators of both apoptosis and autophagy. One of the triggers for mitochondrial mediated apoptosis is the production of reactive oxygen species (ROS). Recently, several studies have indicated that ROS may be also involved in induction of autophagy. ROS are molecules or ions that are formed by the incomplete one-electron reduction of oxygen, including superoxide (O2 (*-)), hydrogen peroxide (H2O2), hydroxyl radical ((*)OH), nitric oxide (NO), and peroxynitrite (ONOO-). Our recent studies provide strong evidences for the involvement of mitochondrially-generated ROS production in the induction of autophagy as determined by the formation of autophagosomes and autolysosomes. This was accomplished through treatment with mitochondrial toxins that inhibit the electron transport chain in transformed and cancer cells. In addition, we have determined that H2O2 and 2-methoxyestradiol (inhibitor of superoxide dismutases and electron transport chain) induce autophagy leading to cell death. In contrast, normal astrocytes fail to induce autophagy following treatment with mitochondrial toxins. Herein, we discuss several important points of our studies and provide a model for mitochondrially-induced autophagic cell death mediated by ROS.     

Participation of autophagy in renal ischemia/reperfusion injury

Renal ischemia-reperfusion (I/R) injury is inevitable in transplantation, and it results in renal tubular epithelial cells undergoing cell death. We observed an increase in autophagosomes in the tubular epithelial cells of I/R-injured mouse models, and in biopsy specimens from human transplanted kidney. However, it remains unclear whether autophagy functions as a protective pathway, or contributes to I/R-induced cell death. Here, we employed the human renal proximal tubular epithelial cell line HK-2 in order to explore the role of autophagy under hypoxia (1% O₂) or activation of reactive oxygen species (500 μM H₂O₂). When compared to normoxic conditions, 48 h of hypoxia slightly increased LC3-labeled autophagic vacuoles and markedly increased LAMP2-labeled lysosomes. We observed similar changes in the mouse IR-injury model. We then assessed autophagic generation and degradation by inhibiting the downstream lysosomal degradation of autophagic vacuoles using lysosomal protease inhibitor. We found that autophagosomes increased markedly under hypoxia in the presence of lysosomal protease inhibitors, thus suggesting that hypoxia induces high turnover of autophagic generation and degradation. Furthermore, inhibition of autophagy significantly inhibited H₂O₂-induced cell death. In conclusion, high turnover of autophagy may lead to autophagic cell death during I/R injury.     

Calcium, mitochondria and oxidative stress in neuronal pathology. Novel aspects of an enduring theme

The interplay among reactive oxygen species (ROS) formation, elevated intracellular calcium concentration and mitochondrial demise is a recurring theme in research focusing on brain pathology, both for acute and chronic neurodegenerative states. However, causality, extent of contribution or the sequence of these events prior to cell death is not yet firmly established. Here we review the role of the alpha-ketoglutarate dehydrogenase complex as a newly identified source of mitochondrial ROS production. Furthermore, based on contemporary reports we examine novel concepts as potential mediators of neuronal injury connecting mitochondria, increased [Ca2+]c and ROS/reactive nitrogen species (RNS) formation; specifically: (a) the possibility that plasmalemmal nonselective cationic channels contribute to the latent [Ca2+]c rise in the context of glutamate-induced delayed calcium deregulation; (b) the likelihood of the involvement of the channels in the phenomenon of 'Ca2+ paradox' that might be implicated in ischemia/reperfusion injury; and (c) how ROS/RNS and mitochondrial status could influence the activity of these channels leading to loss of ionic homeostasis and cell death.     

Zinc pyrithione induces cellular stress signaling and apoptosis in Hep-2 cervical tumor cells: the role of mitochondria and lysosomes

Increased intracellular free zinc concentrations are associated with activation of several stress signaling pathways, specific organelle injury and final cell death. In the present work we examined the involvement of mitochondria and lysosomes and their crosstalk in free zinc-induced cell demise. We report that treatment of cervical tumor Hep-2 cells with zinc pyrithione leads to an early appearance of cytoplasmic zinc-specific foci with corresponding accumulation of zinc first in mitochondria and later in lysosomes. Concomitant with these changes, upregulation of expression of metallothionein II A gene as well as the increased abundance of its protein occurs. Moreover, zinc activates p53 and its dependent genes including Puma and Bax and they contribute to an observed loss of mitochondrial membrane potential and activation of apoptosis. Conversely, lysosomal membrane permeabilization and its promoted cleavage of Bid occurs in a delayed manner in treated cells and their effect on decrease of mitochondrial membrane potential is limited. The use of specific inhibitors as well as siRNA technology suggest a crucial role of MT-IIA in trafficking of free zinc into mitochondria or lysosomes and regulation of apoptotic or necrotic cell demise.     

基于自噬溶酶体形成机制调控缺血性脑卒中后神经元自噬流

脑卒中是由脑血管阻塞或出血引发的急性脑血管病，约84%的临床脑卒中患者由脑缺血引起。研究表明，自噬广泛参与并显著影响脑卒中病理生理进程。自噬是一个将陈旧蛋白质、损伤细胞器及多余胞质组分等呈递给溶酶体进行降解的代谢过程，其包括自噬的激活、自噬体的形成和成熟、自噬体与溶酶体融合、自噬产物在自噬溶酶体内消化和降解等过程。自噬流通常被定义为自噬/溶酶体信号机制。最近发现，自噬流障碍是导致缺血性脑卒中后神经元损伤的重要原因，而在自噬过程中任一步骤发生障碍均可导致自噬流损伤。本文重点对自噬体-溶酶体融合的机制，以及该机制在缺血性脑卒中后发生障碍的致病机理进行详细阐述，以期基于自噬体-溶酶体融合机制对神经元自噬流进行调节，进而诱导缺血性脑卒中后的神经保护。本文可为脑卒中病理机制研究指明方向，为脑卒中治疗探寻新的线索。     

The mitochondrial-lysosomal axis theory of aging: accumulation of damaged mitochondria as a result of imperfect autophagocytosis.

Cellular manifestations of aging are most pronounced in postmitotic cells, such as neurons and cardiac myocytes. Alterations of these cells, which are responsible for essential functions of brain and heart, are particularly important contributors to the overall aging process. Mitochondria and lysosomes of postmitotic cells suffer the most remarkable age-related alterations of all cellular organelles. Many mitochondria undergo enlargement and structural disorganization, while lysosomes, which are normally responsible for mitochondrial turnover, gradually accumulate an undegradable, polymeric, autofluorescent material called lipofuscin, or age pigment. We believe that these changes occur not only due to continuous oxidative stress (causing oxidation of mitochondrial constituents and autophagocytosed material), but also because of the inherent inability of cells to completely remove oxidatively damaged structures (biological 'garbage'). A possible factor limiting the effectiveness of mitochondial turnover is the enlargement of mitochondria which may reflect their impaired fission. Non-autophagocytosed mitochondria undergo further oxidative damage, resulting in decreasing energy production and increasing generation of reactive oxygen species. Damaged, enlarged and functionally disabled mitochondria gradually displace normal ones, which cannot replicate indefinitely because of limited cell volume. Although lipofuscin-loaded lysosomes continue to receive newly synthesized lysosomal enzymes, the pigment is undegradable. Therefore, advanced lipofuscin accumulation may greatly diminish lysosomal degradative capacity by preventing lysosomal enzymes from targeting to functional autophagosomes, further limiting mitochondrial recycling. This interrelated mitochondrial and lysosomal damage irreversibly leads to functional decay and death of postmitotic cells.     

Arachidonic acid promotes glutamate-induced cell death associated with necrosis by 12- lipoxygenase activation in glioma cells

Glutamate induced glutathione (GSH) depletion in C6 rat glioma cells, which resulted in cell death. This cell death seemed to be apoptosis through accumulation of reactive oxygen species (ROS) or hydroperoxides representing cytochrome c release from mitochondria and internucleosomal DNA fragmentation. A significant increase of 12-lipoxygenase enzyme activity was observed in the presence of arachidonic acid (AA) under GSH depletion induced by glutamate. AA promoted the glutamate-induced cell death, which reduced caspase-3 activity and diminished internucleosomal DNA fragmentation. Furthermore, AA reduced intracellular NAD, ATP and membrane potentials, which indicated dysfunction of the mitochondrial membrane. Protease inhibitors such as N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and 3, 4-dichloroisocumarin (DCI) but no Ac-DEVD, a caspase inhibitor, suppressed the glutamate-induced cell death. AA reduced the inhibitory effect of TPCK and DCI on the glutamate-induced cell death. These results suggest that AA promotes cell death by inducing necrosis from caspase-3-independent apoptosis. This might occur through lipid peroxidation initiated by ROS or lipid hydroperoxides generated during GSH depletion in C6 cells.     

Autophagic proteins: New facets of the oxygen paradox

Oxygen (O 2), while essential for aerobic life, can also cause metabolic toxicity through the excess generation of reactive oxygen species (ROS). Pathological changes in ROS production can originate through the partial reduction of O 2 during mitochondrial electron transport, as well as from enzymatic sources. This phenomenon, termed the oxygen paradox, has been implicated in aging and disease, and is especially evident in critical care medicine. Whereas high O 2 concentrations are utilized as a life-sustaining therapeutic for respiratory insufficiency, they in turn can cause acute lung injury. Alveolar epithelial cells represent a primary target of hyperoxia-induced lung injury. Recent studies have indicated that epithelial cells exposed to high O 2 concentrations die by apoptosis, or necrosis, and can also exhibit mixed-phenotypes of cell death (aponecrosis). Autophagy, a cellular homeostatic process responsible for the lysosomal turnover of organelles and proteins, has been implicated as a general response to oxidative stress in cells and tissues. This evolutionarily conserved process is finely regulated by a complex interplay of protein factors. During autophagy, senescent organelles and cellular proteins are sequestered in autophagic vacuoles (autophagosomes) and subsequently targeted to the lysosome, where they are degraded by lysosomal hydrolases, and the breakdown products released for reutilization in anabolic pathways. Autophagy has been implicated as a cell survival mechanism during nutrient-deficiency states, and more generally, as a determinant of cell fate. However, the mechanisms by which autophagy and/or autophagic proteins potentially interact with and/or regulate cell death pathways during high oxygen stress, remain only partially understood.     

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.     

Mitochondrial oxidative burst involved in apoptotic response in oats

Apoptotic cell response in oats is induced by victorin, a host-selective toxin secreted by Cochliobolus victoriae and thought to exert toxicity by inhibiting mitochondrial glycine decarboxylase (GDC) in Pc-2/Vb oats. We examined the role of mitochondria, especially the organelle-derived production of reactive oxygen species (ROS), in the induction of apoptotic cell death. Cytofluorimetric analysis showed that victorin caused mitochondrial deltaPsim breakdown and mitochondrial oxidative burst. Ultrastructural analysis using a cytochemical assay based on the reaction of H2O2 with CeCl3 detected H2O2 eruption at permeability transition pore-like sites on the mitochondrial membrane in oat cells treated with victorin. ROS generation preceded the apoptotic cell responses seen in chromatin condensation and DNA laddering. Both aminoacetonitrile (a specific GDC inhibitor) and antimycin A (a mitochondrial complex III inhibitor) also induced mitochondrial H2O2 eruption, and led to the apoptotic response in oat cells. ROS scavengers such as N-acetyl-l-cysteine and catalase suppressed the mitochondrial oxidative burst and delayed chromatin condensation and DNA laddering in the victorin- or antimycin A-treated leaves. These findings indicate possible involvement of mitochondria, especially mitochondrial-derived ROS generation, as an important regulator in controlling apoptotic cell death in oats.     

Roles of mitophagy and the mitochondrial permeability transition in remodeling of cultured rat hepatocytes

In primary culture, hepatocytes dedifferentiate, and their cytoplasm undergoes remodeling. Here, our aim was to characterize changes of mitochondria during

remodeling. Hepatocytes were cultured 1 to 5 days in complete serum-containing Waymouth’s medium. In rat hepatocytes loaded with MitoTracker Green (MTG),

tetramethylrhodamine methylester (TMRM), and/or LysoTracker Red (LTR), confocal microscopy revealed that mitochondria number and mass decreased by approximately

50% between Day 1 and Day 3 of culture. As mitochondria disappeared, lysosomes/autophagosomes proliferated 5-fold. Decreased mitochondrial content

correlated with (a) decreased cytochrome c oxidase activity and mitochondrial number observed by electron microscopy and (b) a profound decrease of PGC-1α mRNA

expression. By contrast, mtDNA content per cell remained constant from the first to the third day of culture, although ethidium bromide (de novo mtDNA synthesis inhibitor)

caused mtDNA to decrease by half from the first to the third culture day. As mitochondria disappeared, their MTG label moved into LTR-labeled lysosomes, which

was indicative of autophagic degradation. A multiwell fluorescence assay revealed a 2.5-fold increase of autophagy on Day 3 of culture, which was decreased by 3-

methyladenine, an inhibitor of autophagy, and also by cyclosporin A and NIM811, both selective inhibitors of the mitochondrial permeability transition (MPT). These findings

indicate that mitochondrial autophagy (mitophagy) and the MPT underlie mitochondrial remodeling in cultured hepatocytes.