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In the mid-1950s, Sam Clark Jr. of the School of Medicine at Washington University in St. Louis looked through his electron microscope at newborn-mouse kidneys and spotted something he’d never seen before. As he later described it, there appeared to be membrane-bound structures within the cytoplasm of the kidney cells. Intriguingly, these structures seemed to contain altered mitochondria.1
Soon after Clark published his observations, several independent researchers supported his findings. These included the Albert Einstein College of Medicine’s Alex Novikoff, who used the term cytolysome for the structures. “Within these cytolysomes remarkable events are in progress . . .” he and his colleague Edward Essner wrote in 1962.2“Cytoplasm has somehow found its way inside the droplets and is apparently in the process of digestion.”
These were the first descriptions of what is known today as macro-autophagy (hereafter referred to as autophagy). The term is derived from the Greek “auto,” meaning self, and “phagein,” meaning to eat. During autophagy, the cell consumes parts of itself in a regulated manner.
The hallmark of autophagy is the formation of a transient double-membrane structure termed a phagophore. In contrast to secretory transport vesicles, which bud off from an organelle with cargo already enclosed within, the phagophore acquires cargo during its assembly. It may form de novo in the cytoplasm as a free-standing structure, or it may be in contact with an organelle such as the endoplasmic reticulum. The phagophore expands sequentially, providing tremendous flexibility with regard to cargo capacity. As it expands, it sequesters cytoplasmic components, including proteins, lipids, and even entire organelles. Once its payload is secured, the phagophore closes and matures into an autophagosome, with the sequestered cargo now enclosed in the lumen of this compartment. The autophagosome then delivers the cargo, via membrane fusion, to the lytic compartments—vacuoles in fungi and plants and lysosomes in metazoans—for degradation and recycling. It was the autophagosome that captured the attention of Clark and Novikoff more than half a century ago.
TYPES OF AUTOPHAGY
Autophagy can be divided into two broad categories, selective and nonselective, based on the nature of what’s being eaten. The most extensively studied and characterized form of autophagy is macroautophagy, which involves the delivery of cellular components to the lysosome (or vacuole in fungi and plants) via a double membrane–bound structure. Two other forms, beyond the scope of this article, are microautophagy and chaperone-mediated autophagy. During microautophagy, the lysosomal membrane invaginates and sequesters nearby cellular materials for degradation and recycling. Chaperone-mediated autophagy, in contrast, is a specialized protein-degradation process involving dedicated lysosomal transporters.
Today, autophagy is recognized as a critical process for maintaining cellular homeostasis, as well as for responding to stressors, such as nutrient deficiency, which may potentially compromise cell survival. When a cell is exposed to such stressors, autophagy, which occurs constitutively at low levels to balance the constant synthesis of biomolecules, is strongly upregulated. This upregulation increases sequestration and degradation of portions of the cell, releasing macromolecules back into the cytosol to power essential metabolic reactions and generate energy.
The contribution of autophagy to cellular health under both normal and stress conditions implies important physiological and pathological roles for this tightly regulated and precisely orchestrated process. Indeed, autophagy has been found to be instrumental during the course of mammalian development. Additionally, recent research has discovered that autophagy is a critical modulator of a wide range of diseases and disorders. Probing the involvement of autophagy in development and disease is crucial for a more complete understanding of the pathway’s roles, and could have implications for maintaining health or treating disease. While we partially understand its overall morphology and function, information about several steps in this intricate pathway are still emerging.
Mechanisms of autophagy
Several catabolic pathways in the cell break down large molecules. Notably, the conjugation of a small protein called ubiquitin to another cellular protein—often followed by the sequential addition of ubiquitin molecules to generate a polyubiquitin chain—can tag that protein for degradation by the proteasome, resulting in the release of amino acids. (See “The Proteasome: A Powerful Target for Manipulating Protein Levels,” The Scientist, May 2017.) Similar degradation mechanisms exist for other biological polymers such as carbohydrates and lipids.
So what makes autophagy unique? The answer lies in the flexibility of autophagosome size and cargo selection. Autophagy can promote degradation en masse for a large number and variety of substrates, enabling cells to quickly and efficiently generate recycled basic building materials in the face of a wide range of nutritional deficiencies. Additionally, autophagy is the only pathway that is capable of degrading entire organelles, either randomly or in a targeted fashion—a critical process for maintaining homeostasis in the complex landscape of the eukaryotic cell.
Autophagy is tightly regulated to ensure that it is ramped up only when required, and then in a timely manner. The central metabolic sensor of the cell, the TOR complex 1 (TORC1, or MTORC1 in mammals), is sensitive to the availability of amino acids and growth factors, and inhibits autophagy induction when these components are abundant. When cells are starved of these molecules, TORC1/MTORC1 is inactivated, promoting an increase in autophagy. Meanwhile, other molecular regulators monitor cells for the status of various nutrients, such as glucose, or for energy in the form of ATP, and trigger autophagy when such nutrients or metabolites reach critically low levels.
Once autophagy is initiated, several autophagy-related (Atg) proteins act together to coordinate the formation of the phagophore and the subsequent steps of autophagy. Yeast ATG genes were discovered in the 1990s, transforming autophagy research, which had been largely descriptive, to being strongly mechanistic at the molecular level. Experiments using the genetically tractable budding yeast Saccharomyces cerevisiae played a major role in helping scientists decipher the basic mechanism of autophagy. Research in other organisms followed shortly afterward, revealing a remarkable evolutionary conservation in the nature and function of the autophagy machinery from yeast to human. (See illustration here.)
While the overall process of auto-phagy is now somewhat clear to scientists, the field is still hard at work trying to find and fit in many missing pieces of the puzzle. The donor of the membrane for the autophagosome, for example, has not been concretely established. (See “The Enigmatic Membrane,” The Scientist, February 2012.) Similarly, we do not fully understand how phagophore expansion is regulated, or what dictates the frequency of autophagosome generation. Even more questions arise when we consider that many types of autophagy are highly selective, and the initiation and regulation of these processes remain mysterious. Further understanding of these selective pathways is crucial because they are intimately linked to embryonic development, healthy growth, and human disease.
Autophagy in development
In the late 1970s, Richard Lockshin of St. John’s University in New York and colleagues demonstrated that auto-
phagy occurs during insect metamorphosis.3 A decade and a half later, researchers showed that mutant yeast cells defective in autophagy do not sporulate.4 These were the first hints that autophagy may play a role in organismal development, but only relatively recently have researchers begun to elucidate the process’s contributions to cellular differentiation and development in metazoans.
SELF DIGESTION
Over the past 15 years, researchers have demonstrated that nonselective autophagy, which is primarily a starvation response, is also required during early mammalian embryogenesis. Autophagy is likely critical for oocyte reprogramming post-fertilization. Oocytes come loaded with maternally derived mRNAs and proteins that are degraded after an egg fuses with a sperm and the zygote divides into two cells, when the embryo’s own genome takes over. (See “New Techniques Detail Embryos’ First Hours and Days,” The Scientist, December 2017.) This transition from maternal to embryonic control likely depends, at least partially, on autophagy. One piece of evidence for this hypothesis is that homozygous Atg5-knockout mouse embryos derived from oocytes that have been depleted of Atg5 transcripts die somewhere in the four- to eight-cell stage.5
Autophagy is likely also necessary for the fetal-to-neonatal transition that occurs at birth. HomozygousAtg5-null mouse embryos created from unmanipulated oocytes of heterozygous Atg5mothers survive gestation, probably due to the presence of maternally derived ATG5 in the oocyte, but die within one day of delivery.6 Indeed, a large increase in autophagy is observed in certain tissues in mice, including in the heart, shortly after birth. The cause of this increase might be the temporary but severe starvation induced by the termination of placental nourishment; autophagy helps release the amino acids and energy sources necessary for survival during this period of need.
Autophagy has been found to be instrumental during the course of mammalian development.
Selective autophagy, too, plays an important role in post-embryonic development. The oxygen-transporting function of red blood cells depends on the presence of large quantities of the oxygen-binding protein hemoglobin. To meet this demand, precursor reticulocytes must eliminate most organelles as they mature into erythrocytes. Reticulocytes lose mitochondria through mitophagy, a type of selective autophagic degradation. Targeted mitochondrial degradation also occurs during zygotic development in several metazoan species, enabling the elimination of paternal mitochondria so that only maternal mitochondria are inherited.7 In 2017, Beth Levine of the University of Texas Southwestern Medical Center and colleagues identified an inner mitochondrial membrane protein that functions as a novel mitophagy receptor, and showed that this protein plays an essential role in clearing paternal mitochondria in C. elegans.8
These are but a few of autophagy’s suspected roles in normal development. And as organisms mature, this recycling pathway continues to maintain a healthy balance of functional organelles and basic biomacromolecules in the cellular environment. Further elucidating autophagy’s roles, not only at the mechanistic level but also in terms of physiological outcomes, will be important for understanding how this dynamic process regulates metabolic homeostasis in organisms of all ages.
Autophagy gone awry
SELECTIVE AUTOPHAGY
See full infographic: WEB | PDF© N.R.FULLER, SAYO-ART LLCGiven the role of autophagy in physiology, it’s not surprising that dysregulation of the process is tied to a number of pathologies, ranging from infectious diseases to neurodegenerative disorders to cancer. Understanding the relationship between autophagy and disease could be critical to designing effective therapeutic interventions.
The first report of autophagy induction in response to infection was published in 1984, when Yasuko Rikihisa, then at Virginia Tech’s College of Veterinary Medicine, reported that incubating mammalian cells with rickettsiae, bacteria that cause a variety of tick-borne diseases, triggered the formation of autophagosomes.9 However, researchers have only recently begun to understand how the eukaryotic cell uses a type of selective autophagy called xenophagy to destroy invading pathogens. Xenophagy is initiated by the ubiquitination of bacterial surface proteins, which are then recognized by ubiquitin-binding autophagy receptors that promote the sequestration of pathogens by phagophores. (See illustration here.)
In this context, autophagy works in concert with components of the innate immune system, such as toll-like receptors (TLRs). TLRs mediate the initial recognition of pathogens, and subsequent TLR signaling promotes inflammation and may stimulate autophagy, at least in part by modifying the autophagy receptors.10 While inflammation is a necessary step in the recruitment of immune cells to the site of infection, an extended inflammatory response can be detrimental to the tissue. Research from the lab of Shizuo Akira at Osaka University in Japan has revealed that loss of the essential autophagy protein ATG16L1 leads to increased production of proinflammatory cytokines in mice.11This finding suggests that, during the course of the immune response, autophagy not only mediates pathogen elimination but may also limit inflammation to prevent unnecessary tissue damage.
More recently, work from J. Magarian Blander’s laboratory at Weill Cornell Medical College has revealed that autophagy of the endoplasmic reticulum is an essential component of a cellular stress response pathway initiated upon infection with live, Gram-positive bacteria. In this cascade, autophagy plays an instrumental role in mobilizing an efficient innate immune response following a stress response from infected cells, in part by causing the relocalization of the sensor protein TMEM173/STING.12
However, modulating the immune system by autophagy may not always be beneficial. A recent study from Jan Lünemann’s laboratory at the University of Zurich suggested that components of the autophagy machinery might play a role in aggravating multiple sclerosis, an autoimmune disease affecting the central nervous system (CNS). A class of immune cells called dendritic cells use ATG-dependent phagocytosis to present peptides derived from the degradation of myelinated CNS-resident cells as antigens. These self antigens promote the activation of autoreactive effector T cells, leading to an autoimmune attack and progressive nerve degeneration.13
Autophagy’s predominant role, however, is cytoprotective, as is highlighted by its protective function in several neurodegenerative disorders, such as Huntington’s disease (HD) and Parkinson’s disease (PD). David Rubinsztein of the Cambridge Institute for Medical Research and colleagues were among the first to show that autophagy promotes the degradation of aggregation-prone proteins such as mutant HTT (huntingtin), one of the primary culprits in HD.14 They later reported that the activation of autophagy rescues neurodegenerative phenotypes in fly and mouse models of HD.15
In PD, the loss of mitophagy has been identified as a key factor in pathogenesis. Although most PD cases are sporadic, mutations in the mitophagy-related genes PINK1 and PRKN are commonly associated with familial PD. And in 2016, research from the Stanford University School of Medicine laboratory of Xinnan Wang indicated that sporadic cases of PD may also be intimately connected to mitophagy deficiencies.16 This is not unexpected, because neurons heavily depend on mitochondrial ATP synthesis for carrying out energy-intensive processes such as generation and transmission of action potentials, neurotransmitter trafficking, and synaptic signaling. Therefore, mitochondrial homeostasis is critical for proper neuronal function. Neurons use mitophagy to selectively eliminate damaged mitochondria that are not only inefficient at producing ATP, but also generate high levels of reactive oxygen species (ROS), which promote protein and lipid oxidation and DNA damage, causing further cellular dysfunction. The ROS-induced death of mutant mitophagy-deficient neurons is likely a major factor in PD pathogenesis.
Mutations in the mitophagy-related genes PINK1 and PRKN are commonly associated with familial PD.
Autophagy has also been linked to cancer. In 1999, the Levine group made the landmark discovery that mice with only one functional copy of the autophagy-related gene Becn1 exhibit increased tumorigenesis.17 In contrast to the apparent role of autophagy in blocking tumor initiation, however, there is evidence that autophagy can also promote malignancy in established tumors. The duality of autophagy’s role in cancer is partially explained by the factors involved in tumor generation versus progression. Oncogenic transformation is promoted by stress factors such as genomic instability, metabolic disruption, and mitochondrial dysfunction; auto-phagy actively suppresses these stresses and thereby inhibits tumor initiation. However, once a tumor has formed, the playing field radically changes. Cells within the tumor have increased metabolic demands due to faster growth and proliferation rates, but these cancerous cells are not well supplied with nutrients by the vasculature. These cells meet their metabolic needs in part by upregulating autophagy.
Recent research has revealed that the role of autophagy in promoting tumor progression is not limited to cellular effects in the cancer itself, but extends to modulation of the tumor microenvironment. In 2017, Tor Erik Rusten’s group from the University of Oslo demonstrated that inDrosophila melanogaster, increased autophagy within the tumor microenvironment promotes tumor growth and invasion.18 Similarly, work from Alec Kimmelman and colleagues at NYU Langone Health’s Perlmutter Cancer Center has elucidated the interaction between tumor cells of pancreatic ductal adenocarcinoma and stroma-associated pancreatic stellate cells (PSCs). This research suggests that the tumor cells promote autophagy in PSCs, stimulating the production of metabolites and basic biomolecules by these cells. The tumor cells then capture and use these metabolites for their survival and growth under otherwise nutrient-poor conditions.19
Because cancer cells rely on autophagy for survival, inhibiting the process is an intriguing strategy for antitumor therapeutics; however, limiting autophagy can increase susceptibility to infection and neurodegeneration.20 Therapeutically targeting a pathway as critical as autophagy is not straightforward.
Outlook
As a scientific field, the study of autophagy is at a very exciting juncture. Although researchers have partially uncovered some of the mechanisms related to autophagy initiation, progression, and regulation, our understanding of the role of autophagy in development and disease is at an early stage, and there are still more questions than answers concerning the functions of the autophagy-related proteins. Further realization of the role of the process in pathophysiology promises to reveal as yet unanticipated avenues for therapeutic intervention. We already knew that we must eat to live, and it’s now clear that this can sometimes extend to eating ourselves—at least on a cellular level.
Vikramjit Lahiri is a second-year PhD candidate in Professor Daniel J. Klionsky’s laboratory at the University of Michigan in Ann Arbor, where they study the molecular details of autophagy in the budding yeast Saccharomyces cerevisiae.
Article Dated: 01 March 2018
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