Evidence for selective mitochondrial autophagy and failure in aging

Autophagy is a major intracellular degradation/recycling system ubiquitous in eukaryotic cells. It contributes to the turnover of cellular components by delivering portions of the cytoplasm and organelles to lysosomes, where they are digested. Starvation-induced autophagy is required for maintaining an amino acid pool for gluconeogenesis and for the synthesis of proteins essential to survival under starvation conditions. In addition, autophagy plays an important role in the degradation of excess or injured organelles, including mitochondria. To test the hypothesis of an involvement of a decrease in autophagy in the process of aging, we explored the antiaging effects of pharmacological stimulation of autophagy on the age-dependent accumulation of 8-OHdG-rich mitochondria in rat liver. Male 3-month and 16-month-old 24 hours-fasted Sprague Dawley rats were injected with the antilipolytic agent [3,5-dimethylpyrazole (DMP)] intraperitoneally. Results showed that drug injection rescued older cells from the accumulation of 8-OHdG in the mtDNA in less than 6 hours, but no significant decrease in the level of cytochrome c oxidase activity was observed. Together, these data provide indirect evidence that 8-OHdG might accumulate in a small pool of mitochondria with increasing age rather than be degraded by the autophagic machinery selectively.     

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.     

Autophagy determines mtDNA copy number dynamics during starvation

Derived from bacterial ancestors, mitochondria have maintained their own albeit strongly reduced genome, mitochondrial DNA (mtDNA), which encodes for a small and highly specialized set of genes. MtDNA exists in tens to thousands of copies packaged in numerous nucleoprotein complexes, termed nucleoids, distributed throughout the dynamic mitochondrial network. Our understanding of the mechanisms of how cells regulate the copy number of mitochondrial genomes has been limited. Here, we summarize and discuss our recent findings that Mip1/POLG (mitochondrial DNA polymerase gamma) critically controls mtDNA copy number by operating in 2 opposing modes, synthesis and, unexpectedly, degradation of mtDNA, when yeast cells face nutrient starvation. The balance of the 2 modes of Mip1/POLG and thus mtDNA copy number dynamics depends on the integrity of macroautophagy/autophagy, which sustains continuous synthesis and maintenance of mtDNA. In autophagy-deficient cells, a combination of nucleotide insufficiency and elevated mitochondrial ROS production impairs mtDNA synthesis and drives mtDNA degradation by the 3?-5?-exonuclease activity of Mip1/POLG resulting in mitochondrial genome depletion and irreversible respiratory deficiency.

Abbrivations: mtDNA: mitochondrial DNA; mtDCN: mitochondrial DNA copy number.     

Maternal inheritance of mitochondrial DNA

Maternal inheritance of mitochondrial DNA (mtDNA) is generally observed in many eukaryotes. Sperm-derived paternal mitochondria and their mtDNA enter the oocyte cytoplasm upon fertilization and then normally disappear during early embryogenesis. However, the mechanism underlying this clearance of paternal mitochondria has remained largely unknown. Recently, we showed that autophagy is required for the elimination of paternal mitochondria in Caenorhabditis elegans embryos. Shortly after fertilization, autophagosomes are induced locally around the penetrated sperm components. These autophagosomes engulf paternal mitochondria, resulting in their lysosomal degradation during early embryogenesis. In autophagy-defective zygotes, paternal mitochondria and their genomes remain even in the larval stage. Therefore, maternal inheritance of mtDNA is accomplished by autophagic degradation of paternal mitochondria. We also found that another kind of sperm-derived structure, called the membranous organelle, is degraded by zygotic autophagy as well. We thus propose to term this allogeneic (nonself) organelle autophagy as allophagy.     

AoAtg26, a putative sterol glucosyltransferase,is required for autophagic degradation of peroxisomes,mitochondria, and nuclei in the filamentous fungus Aspergillus oryzae

Autophagy is a conserved process in eukaryotic cells for degradation of cellular proteins and organelles. In filamentous fungi, autophagic degradation of organelles such as peroxisomes, mitochondria, and nuclei occurs in basal cells after the prolonged culture, but its mechanism is not well understood. Here, we functionally analyzed the filamentous fungus Aspergillus oryzae AoAtg26, an ortholog of the sterol glucosyltransferase PpAtg26 involved in pexophagy in the yeast Pichia pastoris. Deletion of Aoatg26 caused a severe decrease in conidiation and aerial hyphae formation, which is typically observed in the autophagy-deficient A. oryzae strains. In addition, cup-shaped AoAtg8-positive membrane structures were accumulated in the Aoatg26 deletion strain, indicating that autophagic process is impaired. Indeed, the Aoatg26 deletion strain was defective in the degradation of peroxisomes, mitochondria, and nuclei. Taken together, AoAtg26 plays an important role for autophagic degradation of organelles in A. oryzae, which may physiologically contribute to the differentiation in filamentous fungi.     

Different fates of mitochondria: alternative ways for degradation?

Cellular degradative processes including proteasomal and vacuolar/lysosomal (autophagic) degradation, as well as the activity of proteases (both cytosolic and mitochondrial), provide for a continuous turnover of damaged and obsolete macromolecules and organelles. Mitochondria are organelles essential for respiration and oxidative energy production in aerobic cells; they are also required for multiple biosynthetic pathways. As such, mitochondrial homeostasis is very important for cell survival. We review the evidence regarding the possible mechanisms for mitochondrial degradation. Increasingly, the evidence suggests autophagy plays a central role in the degradation of mitochondria. How mitochondria might be specifically selected for autophagy (mitophagy) remains an open question, although some evidence suggests that, under certain circumstances, in mammalian cells the Mitochondrial Permeability Transition (MPT) plays a role in initiation of the process. As more is learned about the functioning of autophagy as a degradation process, the greater the appreciation we are developing concerning its role in the control of mitochondrial degradation.     

Highlights from this issue

Cellular degradative processes including proteasomal and vacuolar / lysosomal (autophagic) degradation, as well as the activity of proteases (both cytosolic and mitochondrial), provide for a continuous turnover of damaged and obsolete macromolecules and organelles. Mitochondria are organelles essential for respiration and oxidative energy production in aerobic cells; they are also required for multiple biosynthetic pathways. As such, mitochondrial homeostasis is very important for cell survival. We review the evidence regarding the possible mechanisms for mitochondrial degradation. Increasingly, the evidence suggests autophagy plays a central role in the degradation of mitochondria. How mitochondria might be specifically selected for autophagy (mitophagy) remains an open question, although some evidence suggests that, under certain circumstances, in mammalian cells the Mitochondrial Permeability Transition (MPT) plays a role in initiation of the process. As more is learned about the functioning of autophagy as a degradation process, the greater the appreciation we are developing concerning its role in the control of mitochondrial degradation.     

Selective mitochondrial autophagy during erythroid maturation

Accumulating evidence suggests that autophagy can be selective in the clearance of organelles in yeast and in mammalian cells. We have observed that the sequestration of mitochondria by autophagosomes was defective in reticulocytes in the absence of Nix. Nix is required for the dissipation of mitochondrial membrane potential (ΔΨm) during erythroid maturation. Moreover, pharmacological agents that induce the loss of ΔΨm can restore the sequestration of mitochondria by autophagosomes and promote mitochondrial clearance in Nix-/- erythroid cells. Our data suggest that mitochondrial depolarization induces recognition and sequestration of mitochondria by autophagosomes. Elucidating the mechanisms underlying selective mitochondrial autophagy not only will help us to understand the mechanisms for erythroid maturation, but also may provide insights into mitochondrial quality control by autophagy in the protection against aging, cancer, and neurodegenerative diseases.

Addendum to: Sandoval H, Thiagarajan P, Dasgupta SK, Schumacher A, Prchal JT, Chen M, Wang J. Essential role for Nix in autophagic maturation of erythroid cells. Nature 2008; 454:232-5.     

Dynamic Regulation of Autophagy and Endocytosis for Cell Remodeling During Early Development

Fertilization triggers cell remodeling from each gamete to a totipotent zygote. Using Caenorhabditis elegans as a model system, it has been revealed that lysosomal degradation pathways play important roles in cellular remodeling during this developmental transition. Endocytosis and autophagy, two pathways leading to the lysosomes, are highly upregulated during this period. A subset of maternal membrane proteins is selectively endocytosed and degraded in the lysosomes before the first mitotic cell division. Autophagy is also induced shortly after fertilization and executes the degradation of paternally inherited embryonic organelles, e.g. mitochondria and membranous organelles. This mechanism underlies the maternal inheritance of the mitochondrial genome. Autophagy is also required for the removal of extra P‐granule (germ granules in C. elegans) components in somatic cells of early embryos and thereby for the specific distribution of P‐granules to germ cells. This review focuses on recent advances in the study of the physiological roles and mechanisms of lysosomal pathways during early development in C. elegans.      

Autophagy in the pathogenesis of myelodysplastic syndrome and acute myeloid leukemia

Autophagy is a conserved cellular pathway responsible for the sequestration of spent organelles and protein aggregates from the cytoplasm and their delivery into lysosomes for degradation. Autophagy plays an important role in adaptation to starvation, in cell survival, immunity, development and cancer. Recent evidence in mice suggests that autophagic defects in hematopoietic stem cells (HSCs) may be implicated in leukemia. Indeed, mice lacking Atg7 in HSCs develop an atypical myeloproliferation resembling human myelodysplastic syndrome (MDS) progressing to acute myeloid leukemia (AML). Our studies suggest that accumulation of damaged mitochondria and reactive oxygen species result in cell death of the majority of progenitor cells and, possibly, concomitant transformation of some surviving ones. Interestingly, bone marrow cells from MDS patients are characterized by mitochondrial abnormalities and increased cell death. A role for autophagy in the transformation to cancer has been proposed in other cancer types. This review focuses on autophagy in human MDS development and progression to AML within the context of the role of mitochondria, apoptosis and reactive oxygen species (ROS) in its pathogenesis.Key words: autophagy, mitophagy, Atg7, hematopoiesis, HSCs, myelodysplastic syndrome, acute myeloid leukemia     

Localized de novo phospholipid synthesis drives autophagosome biogenesis

ABSTRACT

During (macro)autophagy, cells form transient organelles, termed autophagosomes, to target a broad spectrum of substrates for degradation critical to cellular and organismal health. Driven by rapid membrane assembly, an initially small vesicle (phagophore) elongates into a large cup-shaped structure to engulf substrates within a few minutes in a double-membrane autophagosome. In particular, how autophagic membranes expand has been a longstanding question. Here, we summarize our recent work that delineates a pathway that drives phagophore expansion by localized de novo phospholipid synthesis. Specifically, we found that the conserved acyl-CoA synthetase Faa1 localizes to nucleated phagophores to locally activate fatty acids for de novo phospholipid synthesis in the neighboring ER. These newly synthesized phospholipids are then preferentially incorporated into autophagic membranes and drive the expansion of the phagophore into a functional autophagosome. In summary, our work uncovers molecular principles of how cells coordinate phospholipid synthesis and flux with autophagic membrane formation during autophagy.

Abbreviations: ACS: acyl-CoA synthestases; CoA: coenzyme A; ER: endoplasmic reticulum     

Cytoplasmic inheritance of mitochondria and chloroplasts in the anisogamous brown alga Mutimo cylindricus (Phaeophyceae)

Based on the morphology of gametes, sexual reproduction in brown algae is usually classified into three types: isogamy, anisogamy, and oogamy. In isogamy, chloroplasts and chloroplast DNA (chlDNA) in the sporophyte cells are inherited biparentally, while mitochondria (or mitochondrial DNA, mtDNA) is inherited maternally. In oogamy, chloroplasts and mitochondria are inherited maternally. However, the patterns of mitochondrial and chloroplast inheritance in anisogamy have not been clarified. Here, we examined derivation of mtDNA and chlDNA in the zygotes through strain-specific PCR analysis using primers based on single nucleotide polymorphism in the anisogamous brown alga Mutimo cylindricus. In 20-day-old sporophytes after fertilization, mtDNA and chlDNA derived from female gametes were detected, thus confirming the maternal inheritance of both organelles. Additionally, the behavior of mitochondria and chloroplasts in the zygotes was analyzed by examining the consecutive serial sections using transmission electron microscopy. Male mitochondria were isolated or compartmentalized by a double-membrane and then completely digested into a multivesicular structure 2 h after fertilization. Meanwhile, male chloroplasts with eyespots were observed even in 4-day-old, seven-celled sporophytes. The final fate of male chloroplasts could not be traced. Organelle DNA copy number was also examined in female and male gametes. The DNA copy number per chloroplast and mitochondria in male gametes was lower compared with female organelles. The degree of difference is bigger in mtDNA. Thus, changes in different morphology and DNA amount indicate that maternal inheritance of mitochondria and chloroplasts in this species may be based on different processes and timing after fertilization.

     

Caloric Restriction and Autophagy in Caenorhabditis elegans

Autophagy is a catabolic process in which long-lived proteins and organelles are degraded for recycling in the cytoplasm. In the nematode Caenorhabditis elegans autophagy is associated with formation of the dauer larva, an alternative developmental stage that worms can enter under poor growth conditions. We have shown that C. elegans mutants that experience caloric restriction because they are feeding-defective also exhibit elevated autophagy and decreased levels of fat deposits, as well as smaller cells and, consequently, a smaller body size. Our results suggest novel relationships between caloric restriction, longevity, body size and autophagy.

Addendum to:

C. elegans Feeding Defective Mutants Have Shorter Body Lengths and Increased Autophagy

C. Mörck and M. Pilon

BMC Dev Biol 2006; 6:39     

Mechanisms of mitochondria and autophagy crosstalk

Autophagy is a cellular survival pathway that recycles intracellular components to compensate for nutrient depletion and ensures the appropriate degradation of organelles. Mitochondrial number and health are regulated by mitophagy, a process by which excessive or damaged mitochondria are subjected to autophagic degradation. Autophagy is thus a key determinant for mitochondrial health and proper cell function. Mitophagic malfunction has been recently proposed to contribute to progressive neuronal loss in Parkinson disease. In addition to autophagy''s significance in mitochondrial integrity, several lines of evidence suggest that mitochondria can also substantially influence the autophagic process. The mitochondria''s ability to influence and be influenced by autophagy places both elements (mitochondria and autophagy) in a unique position where defects in one or the other system could increase the risk to various metabolic and autophagic related diseases.Key words: autophagy, mitochondria, fission, fusion, apoptosis     

Multivesicular Bodies and Autophagy in Erythrocyte Maturation

During reticulocyte maturation, hematopoietic progenitors undergo numerous changes to reach the final functional stage which concludes with the release of reticulocytes and erythrocytes into circulation. During this process some proteins, which are not required in the mature stage, are sequestered in the internal vesicles present in multivesicular bodies (MVBs). These small vesicles are known as exosomes because they are released into the extracellular medium by fusion of the MVB with the plasma membrane. Interestingly, during this maturation process some organelles, such as mitochondria and endoplasmic reticulum, are wrapped in double membrane vacuoles and degraded via autophagy. We have demonstrated in human leukemic K562 cells a role for calcium and Rab11 in the biogenesis of MVBs and exosome release. Here we discuss evidence indicating that K562 cells present a high basal level of autophagy, and that there is an association between MVBs and autophagosomes, suggesting a role for the autophagic pathway in the maturation process of this cell type.

Addendum to:

Exosome secretion and red cell maturation: Exploring molecular components involved in the docking and fusion of multivesicular bodies in K562 cells.

Fader CM, Savina A, Sánchez D, Colombo MI. Blood Cells Mol Dis 2005; 35:153-7.

and

Rab11 promotes docking and fusion of multivesicular bodies in a calcium-dependent manner.

Savina A, Fader CM, Damiani MT, Colombo MI. Traffic 2005; 6:131-43.     

Maternal inheritance of mitochondrial DNA: Degradation of paternal mitochondria by allogeneic organelle autophagy, allophagy

Maternal inheritance of mitochondrial DNA (mtDNA) is generally observed in many eukaryotes. Sperm-derived paternal mitochondria and their mtDNA enter the oocyte cytoplasm upon fertilization and then normally disappear during early embryogenesis. However, the mechanism underlying this clearance of paternal mitochondria has remained largely unknown. Recently, we showed that autophagy is required for the elimination of paternal mitochondria in Caenorhabditis elegans embryos. Shortly after fertilization, autophagosomes are induced locally around the penetrated sperm components. These autophagosomes engulf paternal mitochondria, resulting in their lysosomal degradation during early embryogenesis. In autophagy-defective zygotes, paternal mitochondria and their genomes remain even in the larval stage. Therefore, maternal inheritance of mtDNA is accomplished by autophagic degradation of paternal mitochondria. We also found that another kind of sperm-derived structure, called the membranous organelle, is degraded by zygotic autophagy as well. We thus propose to term this allogeneic (nonself) organelle autophagy as allophagy.     

Autophagosome-Lysosome Fusion Depends on the pH in Acidic Compartments in CHO Cells

Autophagy is the bulk degradation of cytoplasmic constituents in response to starvation and other environmental or intracellular cues. During this process, most of the cytoplasm is sequestered into autophagosomes, which then fuse with lysosomes where the degradation of the sequestered material proceeds. We investigated the relationship between autophagosome-lysosome fusion and the pH in acidic compartments by visualizing the fusion process using fluorescence in CHO cells. In this experiment, mitochondria were labeled with GFP by transfecting CHO cells with the presequence of ornithine transcarbamylase, and lysosomes were labeled with Texas Red Dextran; any fusion was identified by the colocalization of mitochondria (in autophagosomes) and lysosomes using fluorescence microscopy. When CHO cells were treated with rapamycin or starvation medium to induce autophagy, the colocalization of fluorescence was observed. Whereas when they were treated with 3-MA, an inhibitor of autophagy, the colocalization disappeared. We conclude that the colocalization reflects the fusion of autophagosomes and lysosomes. Moreover, when the CHO cells were treated with drugs that increase the pH of acidic compartments, the colocalization disappeared. This suggests that the autophagosome-lysosome fusion is inhibited by increasing pH in acidic compartments independently of V-ATPase activity in CHO cells.

Addendum to:

Quantitative Monitoring of Autophagic Degradation

Akinori Kawai, Syuichi Takano, Nobuhiro Nakamura and Shoji Ohkuma

Biochem Biophys Res Commun 2006; 351:71-7