Longevity

Courtesy Edward T.W. Bampton, Gerry Smith, Alena Pance During autophagy-literally "self-eating"-cells deliver cytoplasmic constituents, including whole organelles, to the lysosome for degradation. This crucial recycling process kicks in during gross developmental changes and times of nutrient deprivation. New work may place it within cellular aging pathways as well. Characterized morphologically by Christian de Duve in the 1960s, autophagy was a natural extension of his Nobel Prize-winning work on lysosomes. Veteran researchers in a once obscure field point to the discovery of the molecular machinery a decade ago-autophagy gene products that drive the process-as a boost to their own work. More recently, the linkage of one of these genes with tumorigenesis placed autophagy under a new spotlight and is credited with widening the investigative pool. Now scientists are finding evidence-both circumstantial and causal-that autophagy is a key mechanism in how certain manipulations, namely mutations in insulin signaling and caloric restriction, promote longevity. Autophagy often gets overlooked as "just housekeeping," says Beth Levine, professor of internal medicine at the University of Texas-Southwestern Medical Center at Dallas. In fact, she says, failures in keeping house likely contribute to diseases such as cancer and neurodegeneration. In addition, autophagy wanes with age for reasons that aren't yet clear, says Levine, and is "mechanistically important" in aging itself. FROM FEAST TO FAMINE When food is scarce, autophagy gets turned on as a survival mechanism. When food is moderately restricted for a long time, organisms from worms to mammals live longer. Ettore Bergamini, professor of pathology at the University of Pisa, Italy, provides some evidence that increased autophagy may accompany calorie-restricted life extension. In rats subject to caloric restriction, autophagic efficiency is maintained in old age and rats live longer.1 "The problem of aging is free radical production and imperfection of the cell-repair mechanisms," says Bergamini. Perhaps more general contractor than housekeeper, autophagy is a "second-line defense" against the maintenance challenges of cells. "One hundred million free radicals are generated per day in our body," he says. "Ten thousand DNA lesions per day per cell are produced." Defects that slip past the front-line defenses-DNA repair systems and proteosomal degradation-are refurbished, and damaged organelles are removed. Also, if autophagy falls off in age, then the detritus of cell metabolism accumulate more readily. By degrading damaged organelles that generate reactive oxygen species, autophagy fits nicely into the mitochondrial theory of aging. The capacity of autophagy to catabolize whole organelles, and mitochondria in particular, may be its strongest selling point in terms of being an antiaging mechanism. Mitochondria are a frequent target of autophagy, says John Lemasters, professor of cell and developmental biology at the University of North Carolina. "They will sort of wear out," he says. "In nonproliferating tissues like brain, heart, and liver, the mitochondria turn over every 10 to 25 days." This rate is accelerated by nutrient deprivation and glucagon, both stimulants of autophagy. Lemasters studies specific changes in damaged mitochondria that may invite their degradation. "Autophagy becomes a mechanism to remove the ones that ought to be eliminated," he says. Damaged mitochondria might generate even more free radicals to "do mischief," which can be especially treacherous in nondividing tissues like heart and brain "where age-dependent effects really manifest themselves quite strongly." Age-related diseases of these tissues-cardiomyopathy and neurodegeneration-have been implicated as disorders of autophagy. MOLECULAR MACHINERY The term autophagy generally refers to macroautophagy, in which an intracellular membrane forms, encircles a chunk of cytoplasm, and subsequently fuses with the lysosome where enzymatic degradation takes place. Other autophagic processes occur directly at the lysosomal membrane either via direct engulfment of cytosol (microautophagy) or via chaperone-mediated docking of damaged proteins. Autophagy is upregulated in times of stress or change. By breaking down large proteins and organelles and feeding spare parts into protein-synthesis and energy-production pathways, the process allows cells to be temporarily self-sustaining during starvation. Another trigger is developmental tissue remodeling, such as insect metamorphosis, where organisms are "not only making new structures, but getting rid of old ones," says Daniel Klionsky, professor of biological chemistry at the University of Michigan's Life Sciences Institute. Researchers are studying the molecular regulation intensely. And by depleting nutrients, researchers can watch autophagy unfold. AUTOPHAGY IN ACCLIMATION © 2004 Nature Publishing Group In transgenic mice expressing GFP fused to the autophagy protein LC3, autophagosomes are absent in embryonic heart tissue. They can be easily found after birth, (0.5 hours postnatal), but begin to wane at 6 hours and are largely absent 2.5 days postnatal. (From A. Kuma et al., Nature, 432:1032–6, 2004.) The most-studied step is the elongation of membranes derived from endoplasmic reticulum to form the autophagosome. "The autophagosome is essentially made on demand," says Klionsky. Morphological evidence has long indicated that autophagy is a highly conserved mechanism. And while the genes were initially discovered in yeast, analogs of those genes are now being found in worms, flies, and mammals. Assembly of the autophagosome requires the interaction of two autophagy proteins, designated Atg1 and Atg13, says Ana Maria Cuervo, assistant professor of anatomy and structural biology at the Albert Einstein College of Medicine in New York. "The major cascade that is involved is insulin modulating the phosphorylation of Atg proteins." When Atg1 and Atg13 are hyperphosphorylated, they cannot interact and elongation is blocked. A kinase named target of rapamycin (TOR) controls the phosphorylation of the Atg proteins, says Cuervo. With significant roles in cell cycle,2 it's now believed to be one of the key negative regulators for autophagy. Says Klionsky, "When you are starved, TOR is inactivated and that allows autophagy to be induced.""TOR responds to two well-characterized inputs," says Thomas Neufeld, assistant professor of genetics, cell biology, and development at the University of Minnesota. When insulin binds its receptor in the plasma membrane, it activates a signal transduction cascade beginning with phosphoinositide 3-kinase (PI3K) and converging on the GTPase Rheb. "Rheb is probably the most immediate known upstream activator of TOR," Neufeld says. The second pathway doesn't involve insulin signaling but allows TOR to respond "more directly" to nutrient levels in a cell. "Starvation of nutrients doesn't affect PI3K, but dramatically affects TOR," says Neufeld. It's likely the older pathway, preserved from primitive single-celled organisms. "That's the way it seems to work in yeast, which doesn't have an insulin signaling pathway.""The beauty of this autophagy system is it's completely conserved from yeast to humans," says Harald Stenmark, professor of biochemistry at the Norwegian Radium Hospital in Oslo, Norway. "So you can find basically all the components of the autophagic machinery in yeast in humans as well." LINKS TO LONGEVITY SURVIVING BIRTH The journey from uterus to the outside world involves more than navigating the birth canal. Neonates must transition from a liquid environment to air. At the same time, they are abruptly deprived of their maternal pipeline of nutrients and must learn to nurse. A recent report portrays autophagy as wearing two hats during this critical time, working both as a developmental manager and as a nutrient provider.1 Since its first description in the 1960s, autophagy has been visualized using electron microscopy, a laborious and time-consuming method. Now, Japanese researchers have developed a quick and specific method by tagging an autophagy protein-LC3, the mammalian homolog of Atg8-with green fluorescent protein, which allows visualization of autophagosomes with fluorescent microscopy. Using this technique, Noboru Mizushima and colleagues from the National Institute for Basic Biology in Okazaki, Japan, observed that while autophagy is generally low in utero, it is rapidly upregulated at birth. After a day or two, it returns to basal levels. The magnitude of autophagy induction varied according to tissue and differed from the pattern seen in adult mice subject to starvation, prompting the authors of the study to speculate on their significance. Heart and diaphragm showed large increases in autophagosome number, perhaps due to the increased energy demands of those muscles as the newborn takes over control of those functions. Alveolar and skin cells also showed large increases, implicating a developmental response to the drastic change in environment. To test whether autophagy is necessary to survive transient starvation after birth, researchers made knockout mice lacking one of the genes necessary for autophagy, Atg5. Knockout mice appeared normal at birth, but showed severe nutrient/energy crisis soon after and died at twice the rate as normal mice when food was withheld. Thus, autophagy provides the substrates or energy sources needed to bridge the transition from a placenta-fed fetus to a milk-fed infant. "The role of autophagy during the early neonatal starvation period," Kuma A, Nature Vol 432, 1032-6 Dec. 23, 2004 In nematodes, researchers have linked autophagy genes to experimentally increased lifespan. Caenorhabditis elegans with a mutation in daf-2 live nearly twice as long as wild-type animals. Daf-2 normally encodes the insulin/insulin-like growth factor receptor. Inhibiting the autophagy gene bec-1 shortened the life of the mutants.3 "That's the only primary data so far" linking autophagy genes to longevity, says Levine, a coauthor on the study. Genetic studies in fruit flies from Neufeld4 and Stenmark5 also have provided strong genetic evidence for insulin signaling negatively regulating autophagy, says Levine. "I think it's exciting because I think autophagy is potentially a unifying mechanism, which explains the diverse effects of the signaling pathway regulating cancer and aging." RECYCLING IN TRYING TIMES: © 2005 Elsevier Mammalian autophagy is a cellular defense against two forms of nutrient stress, birth and growth factor deprivation. (from B. Levine, Cell, 120:159–62, 2005.) Cynthia Kenyon, professor of biochemistry and biophysics at the University of California, San Francisco, is not convinced. Kenyon, who first reported the doubling of lifespan in daf-2 mutants, has since studied the altered expression of genes downstream from daf-2.6 The 50 genes that changed the most "fell into many different classes: antioxidant genes, metabolic genes, antibacterial genes," she says. "So it's certainly not only autophagy." Other pathways have certainly been implicated in the lifespan extension induced by calorie restriction. Fasting upregulates expression of sir-2 in yeast and worms, and SIRT-2 in mice, and the resulting proteins may help mobilize glucose for use in the cell.7 At present, no one has tried to link autophagy to the sir-2 pathway, although Levine admits that she's pondered the notion. Aging is ill defined and certainly multifactorial. The appeal of autophagy to ameliorate normal aging is its capacity as a jack-of-all-trades repair mechanism in the cell. "I believe that the induction of autophagy in caloric restriction-or loss-of-function mutation in insulin signaling-leads to increased degradation of damaged mitochondria and reduction of oxidative stress," says Levine. "And that's probably a downstream pathway that's in common to all these life extension phenotypes." REGULATORY MODEL: Courtesy Dan Klionsky In yeast, the Tor kinase exerts a negative regulatory effect on autophagy when cells are growing under nutrient-rich conditions. Most of the proteins required for autophagy are constitutively expressed. When starvation occurs, the Tor kinase is inactivated allowing other downstream effectors, most likely phosphatases and kinases, to modulate such proteins as Atg13 which in turn modulates the key kinase Atg1, required for autophagy. Autophagy has not been a target in aging research for long. And while Klionsky appreciates the attention autophagy is receiving now, he doesn't discount the value of working "in the shadows" before. As one of only a few labs working in yeast, he recounts, "We were going along, making a lot of progress, getting the genes identified." Then Levine's 1999 paper came out implicating autophagy in tumor suppression.8 The response was a flurry of work that Klionsky jokingly compares to party crashers eating all the food and leaving a mess. "Since then, people have solidified the cancer connection," he says. At present, autophagy is implicated in controlling neurodegeneration, myopathy, pathogen invasion, and lifespan. Klionsky adds, "Now you're seeing papers coming out in Science, Nature, and Cell."

Longevity

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Jill Adams

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May 2005

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