In one of the seventh-floor laboratories in an old building at the Federal University of São Paulo (Unifesp), medical student Vitor Neves Sato peers through a microscope every day at lunch time and in the late afternoon to observe the movement of transparent worms known as Caenorhabditis elegans. Never more than one millimeter long, the graceful worms slither tirelessly across a Petri dish like tiny little snakes. Sato is watching to see how a particular antibiotic affects the lifespan of this organism, whose inauguration into laboratory use exactly 50 years ago has afforded a series of major discoveries. It was in C. elegans that biologists identified the first age-related genes and the mechanism of programmed cell death, which is essential to the development of any living being.
Sato has found that the worms that are grown in the antibiotic-rich culture live 9% to 19% longer than those grown without the drug; this is up to ten days more, a considerable length of time for a creature that rarely lives longer than a month. The drug is meant to prolong the lifespan of the worm not by killing bacteria – which C. elegans actually lives off – but by boosting the expression and activity of the enzyme Dicer and of small molecules known as microRNAs (miRNAs). Originally identified in 1993 in C. elegans, miRNAs are a type of ribonucleic acid (RNA) that in this case binds to messenger RNA (mRNA) and helps decrease protein production. According to Marcelo Mori, the professor at Unifesp who coordinates the study, cells should respond to this inhibition by optimizing energy generation and averting the formation of excess waste and residue that can damage DNA and accelerate the development of age-related complications and diseases like cancer.
The antibiotic, whose name Mori is keeping confidential, simulates the effects of caloric restriction, which is a known way of extending lifespan – albeit not feasible for human beings, since it implies cutting food intake by 30% to 40%, “until reaching death,” Mori emphasizes. Another way to achieve the same thing is to inhibit the activity of the mTOR (mammalian target of rapamycin) gene, which plays a role in protein synthesis. In a paper published in August 2013 in Cell Reports, researchers in the United States reduced mTOR gene expression and thereby prolonged the lifespan of mice by about 20%, which is tantamount to lengthening the life of a human being by 16 years. Strangely enough, the longevity of certain tissues and organs varied in this experiment. The animals did better in memory retention, motor coordination, and muscle strength as they aged, but their bones deteriorated faster than normal and they were more susceptible to infection.
Ivan José Vechetti Junior, of the Universidade Estadual Paulista (Unesp) in Botucatu, works simultaneously with miRNA, mTOR, and one effect of aging, which is the loss of muscle mass. In the experiments that he conducted from January through May of 2013 at the University of Kentucky, Vechetti found that expression of a certain type of miRNA, that is, microRNA-1, dropped by half in mice presenting muscle hypertrophy in their paws, caused by removing part of their muscles. Yet in cell cultures, microRNA-1 behaved in a surprising fashion. “MicroRNA-1 was regulated by mTOR in a subtle way, not by increasing its expression, as we had expected, but by prolonging its activity,” says Vechetti.
For his doctorate, under the advisorship of Maeli Dal Pai, Vechetti is investigating miRNA expression in muscle recovery in older rats subjected to immobilization-induced muscular atrophy. If his work goes well, he hopes to find new strategies for attenuating or inhibiting age-related muscle loss – for example, ways to recover lost muscle mass in elderly people who suffer a fall and have to spend some time immobilized. “What are the limits of muscle recovery? How much does physical exercise really help with this recovery?” are questions he poses himself.
The Quasimodo Effect
Research currently underway at Unifesp points to another result of increased miRNA expression: decreased aggregation of the amino acid glutamine. According to Mori, aging, along with illnesses like the neurodegenerative genetic disorder called Huntington’s disease, are associated with the formation of glutamine-rich protein aggregates. To illustrate how he might be on the path to a solution for this problem, Mori points to the C. elegans that were grown in an antibiotic-rich culture and observed this time by Ana Forti Pinca, a biomedical student. Viewed under a microscope, the tiny worms display a number of green spots scattered across their bodies; these are polyglutamines that have bonded to a fluorescent green protein and that help identify the molecules of interest, which move about incessantly. In a petri dish containing worms that did not receive a bath in the antibiotic, the green spheres are smaller but apparently more insoluble, and they seem to hamper movement, bringing to mind Quasimodo, the character in The Hunchback of Notre Dame.
During his post-doctoral studies at Harvard, Mori first used mice to ascertain that one of the main sources of miRNAs is the adipose tissue formed by fat cells, which in the case of humans is usually concentrated under the skin and in the abdominal region. His research suggests that fat cells play an active role in controlling both weight and metabolism rather than simply reflecting the consequences of overeating or of a sedentary lifestyle (see article on page 46). “Adipose tissue acts as a nutritional thermostat,” he explains. “It’s the first to respond when food is restricted, by consuming energy reserves and signaling to muscle cells and other tissues that it’s time to become more efficient.”
It is a known fact that fat cells produce the hormone leptin and can suppress the appetite while stimulating cell metabolism, occasioning weight loss. According to Mori, leptin signaling is in turn linked to aging and to the onset of cardiovascular disease, diabetes, and cancer. Mori’s research describes a reverse situation, in which miRNAs slow down cell activity. At Harvard, Mori measured the amount of miRNAs in the fat tissue of different tissues of younger and older mice. He noted that aging was accompanied by a drop in the amount of miRNA in fat tissues but that caloric restriction reversed this, holding levels of the enzyme Dicer and of miRNAs steady.
The association seemed to be direct, and this was confirmed shortly thereafter in C. elegans: animals with higher levels of Dicer and miRNAs lived longer, while those with lower levels died earlier. “Caloric restriction causes overexpression of Dicer and enhances resistance to oxidative stress, which can damage cells,” Mori pointed out. Furthermore, as described in depth in an article published in the journal Cell Metabolism in 2012, this enzyme’s loss-of-function mutations prompted cellular senescence. “MiRNA expression in adipose tissue can control aging,” he concluded.
Mori began working with C. elegans at Harvard in 2007 because he needed organisms with a shorter life cycle than mice. In 1963, South African biologist Sydney Brenner, originally at Cambridge, England, and later at San Diego, likewise initiated research with C. elegans, because he was looking for an organism that would not only grow fast but that would also allow him to observe cell and organ growth, something that was not possible with fruit flies, the classic insect in genetics research.
At first, when Brenner was preparing and selecting the mutants that were to prove vital in studies that would begin breaking ground soon after, most people did not take the worm seriously. A colleague of Brenner’s said he “wouldn’t give him a penny” for his work and lamented the fact that Brenner was 20 years ahead of his time – this is what the biologist reported in 2009, seven years after being named a Nobel recipient in recognition of his research on gene regulation, conducted on C. elegans. “I’ve been told that today there are 400 C. elegans laboratories,” he stated with satisfaction. “I’m often asked why I abandoned work with C. elegans right when it was getting really interesting. The answer is simple: the people in this area today are much better than I was.” What Brenner really liked was blazing trails, which, in his words, was “the most exciting part of scientific research.”
“Only a few people work with C. elegans in Brazil, perhaps because they are leery about how a different experimental model might be accepted, while I saw this as an opportunity,” says Mori, who brought the animals back in his baggage when he returned from the United States two years ago. He now has a collection of 50 lineages, which are kept in four incubators at an average temperature of 21ºC.
So far, there are no applications for his work, because miRNAs actually constitute a group of hundreds of molecules that average 20 nucleotides, and some probably counteract aging while others favor it. The problem is that miRNAs seem to be all over the place and to play many roles. They can, for instance, influence the progression of prostate tumors, as shown by researchers at the University of São Paulo School of Medicine (FMUSP); affect cardiovascular disease, which is being studied at the USP Biomedical Sciences Institute; or have a part in controlling the circadian rhythm, the roughly 24-hour period upon which the biological cycle of nearly every living being is based, as proposed by a group at the Federal University of Alagoas.
Despite uncertainties about the role of these molecules, Mori believes that controlling the enzyme Dicer and miRNAs can offer a viable strategy for prolonging lifespan by simulating the effect of caloric restriction through the application of drugs, like the antibiotic that he is now evaluating. Aging is, of course, a highly complex biological process. At a conference held in Italy last August, Mori noticed that one of the current centers of attention are mitochondria, which are the cell compartments responsible for generating energy. “Communication between the mitrochondrion and the nucleus regulates protein synthesis and in this way controls aging,” he says. “The time is coming when we’ll be able to pull independent information together and have a clearer notion of how the body ages and how we can effectively intervene.”
Identification of mechanisms responsible for the beneficial effects of caloric restriction (10/52557-0); Grant Mechanism Young Investigators Grant Program; Coordinator Marcelo Alves da Silva Mori – Unifesp; Investment R$696,496.53 (FAPESP).
BRENNER, S. In the beginning was the worm… Genetics. v. 182, pp. 413-5, 2009.
MORI, M.A. et al. Role of microRNA processing in adipose tissue in stress defense and longevity. Cell Metabolism. v. 5, no. 16, pp. 336-47, 2012.