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MIT biologists have advanced their research into mechanisms of human aging by determining the subcellular home of WRN, the protein defective in Werner syndrome, a disease which causes symptoms of premature aging.
Absence of or defects in the WRN protein have been shown to cause accelerated aging in human cells. People with Werner syndrome suffer cataracts, osteoporosis, diabetes and cancer at an early age. They succumb to cardiovascular disease or cancer in their forties or fifties.
The WRN gene product is located in the nucleolus, a dense body within the nucleus of a cell. A defective nucleolus may relate to the symptoms of Werner's individuals, according to Professor Leonard Guarente of the Department of Biology.
The research was reported in the June 9 Proceedings of the National Academy of Sciences. The authors are Robert A. Marciniak, David Lombard, F. Bradley Johnson and Professor Guarente.
By unequivocally locating the WRN protein in the nucleolus, the researchers have moved one step closer to their goal of understanding and perhaps one day intervening in the normal aging process for people.
"This phase of research is a stepping-stone. The most exciting possibility is to discover, very specifically, how the nucleolus changes in humans and especially to identify what changes with age. Then we can start to think about ways to slow it down," Professor Guarente said.
A related and significant advance in the same research revealed the subcellular localization of the mouse homologue, or version of WRN (mWRN).
"In contrast to human WRN protein, mWRN protein is present diffusely throughout the nucleus," the authors wrote. "Understanding the function of WRN in these organisms of vastly differing lifespan may yield new insights into the mechanisms of lifespan determination."
The average person lives 80 years; the average mouse, two years. Mice more often die of cancer than old age, so the role of mWRN in their brief lives offers rich possibilities for comparative study.
The most recently reported research evolved from just over a year of intensive publishing and more than seven years' work in the Guarente lab at MIT and other Boston-area facilities.
Professor Guarente recalled the "very MIT-style" beginning of his lab's groundbreaking research.
"I was discussing areas for research with graduate students, looking for the ones that were known to be hard, maybe impossible," he said. "Aging was the area those students chose. I gave them a year to work on it. We didn't get anywhere then, but at the end of the year, we were so fascinated, we just kept going."
After four or five years, the first group accomplished their goals, Professor Guarente said. "Now, a new generation is carrying the ball."
The PNAS article is the fourth to be published on Guarente's work in just over a year. Together, the articles describe the steps leading from discoveries about aging and death in yeast cells to localization of WRN protein in humans and in mice.
In May 1997, an article published in Cell demonstrated that certain yeast genes determine the life span in yeast and showed that those same yeast genes promote cell longevity by moving from one cell structure to another (from the telomeres to the nucleolus).
The nucleolus, of course, would later be recognized as the site where the clock of cell mortality visibly goes tick-tock.
In August 1997, an article in Science written by Dr. David Sinclair and Professor Guarente identified the crucial role of another specific yeast gene, SGS1, in determining the life span of yeast cells. SGS1 corresponds structurally to the human gene, WRN. The MIT biologists discovered that experimental mutation of SGS1 produced symptoms of aging in yeast cells. The main symptoms noted by the researchers were fragmentation and enlargement of the nucleolus.
"In a striking parallel to Werner syndrome in humans, the sgs1 mutation shortens yeast lifespan by approximately 60 percent," the biologists wrote in the PNAS article, summarizing the earlier work.
The research published last August suggested "the nucleolus may be the Achilles' heel as cells get old. We think fragmentation of the nucleolus is a cause of aging," Professor Guarente commented at the time.
Four months later, in Cell (December 26, 1997), co-authors Drs. Sinclair and Guarente reported they had identified the mechanism of enlargement and fragmentation -- in short, the mechanism of aging itself -- in yeast cells.
FROM YEAST TO HUMAN CELLS
The new research "links us back to humans. The major point is that the human protein WRN is localized in the nucleolus. So, for people with Werner syndrome, the problem may lie in the nucleolus," said Professor Guarente.
"Now, the question is, what specific defect in the nucleolus might result in the disease of rapid aging?" he said.
In addition to localizing the WRN protein in normal cell lines, the researchers showed that the marked concentration of WRN persisted in the nucleolus in a variety of normal and cancerous human cells. Thus, the presence of other diseases did not disrupt WRN protein from its appointed rounds.
Once the scientists knew where WRN protein was localized, they again needed to explore what it did. This type of research progresses more like a sailboat tacking into the wind than like a train: the biologists must move patiently and creatively, back and forth across species, to get results.
Yeast cells continue to provide a simpler version of the molecular events occurring within human cells, Professor Guarente said. But the mirror that yeast provides for human cells may err by oversimplifying, too.
"Now we have to ask, is the structural change in human cells analogous to what happens in yeast? In yeast, there is only one such protein (sgs1). In mammals, there are several," he said.
To expand their research and to bridge the gap between the revelatory but simple one-protein yeast cell and the vastly more complex human, the biologists next looked for something a little higher up -- but not too high up -- on the food chain, Professor Guarente said.
They chose the mouse, which displays an intriguingly different localization for mWRN, the mouse equivalent of human WRN protein. What's more, since mice don't seem to age but instead often die of cancer, this difference promises more discoveries, the PNAS article stated.
To gain the most from the difference between mice and men, the researchers first established a link beyond mortality between the two species by finding the mouse equivalent of the WRN gene.
Next, the biologists contrasted immunofluorescent images of WRN and mWRN with surprising and significant results. Images of WRN reveal its presence in the human nucleolus and not elsewhere in the nucleus. WRN appears as a dramatic spot of light, illuminating the nucleolus like a neon sign on a dark country road.
By contrast, mWRN is present diffusely throughout the entire nucleus, so its image in a mouse cell leaves an all-over powdery glow, like new snow.
Thus, the biologists wrote, "mWRN does not show nucleolar localization. It remains to be determined whether this apparent difference in subcellular localization implies a difference in the function of WRN in these two organisms."
The next steps for the biologists include creating and studying "knock-out mice" -- mice without mWRN -- to see if their lives are shortened, as Werner syndrome sufferers' are, by absent or defective WRN genes.
"Such cross-species analysis may aid in our understanding of the importance of nucleolar structure and function in mammalian aging and yield new insights into mechanisms of lifespan determination," the article said.
As for the knockout mice themelves, Professor Guarente added, there were "a few possibilities. We could get normal mice. We could get dead mice. Or, most interesting of all, we could get a mouse that's in between -- a mouse with premature aging."
Implications for future research arising from the phase reported in the recent PNAS article include close study of yeast, mice, and human cells to clarify both nucleolar function and the role of WRN and its homologues in cell aging and death.
This work was supported by grants from the National Institutes of Health to Dr. Marciniak, Johnson and Guarente and a Medical Scientist Training Program Training Grant to David Lombard.
A version of this article appeared in MIT Tech Talk on July 15, 1998.