Lifespan

Youth → broken DNA → genome instability → disruption of DNA packaging and gene regulation (the epigenome) → loss of cell identity → cellular senescence → disease → death.

The implications were profound: if we could intervene in any of these steps, we might help people live longer.

But what if we could intervene in all of them? Could we stop aging?

Theories must be tested and tested and tested some more—not just by one scientist but by many. And to that end, I was fortunate to have been put onto a research team that included some of the most brilliant and insightful scientists in the world.

There was Lenny Guarente, our indefatigable mentor. There was also Brian Kennedy, who started the yeast-aging project in Lenny’s lab and has since played a tremendously important role in understanding premature aging diseases and the impact of genes and molecules that increase health and longevity in model organisms. There were Monica Gotta and Susan Gasser at the University of Geneva, who are now some of the most influential researchers in the field of gene regulation; Shin-ichiro Imai, now a professor at Washington University, who discovered that sirtuins are NAD-utilizing enzymes and now does research into how the body controls sirtuins; Kevin Mills, who ran a lab in Maine, then became a cofounder of and chief scientific officer at Cyteir Therapeutics, which develops novel ways to fight cancer and autoimmune diseases; Nicanor Austriaco, who started the project with Brian, now teacher of biology and theology at Providence College, a great combo; Tod Smeal, chief scientific officer of cancer biology at the global pharmaceutical company Eli Lilly; David Lombard, who is now a researcher in the field of aging at the University of Michigan; Matt Kaeberlein, a professor at the University of Washington, who is testing molecules on dog longevity; David McNabb, whose lab at the University of Arkansas has made key and lifesaving discoveries about fungal pathogens; Bradley Johnson, an expert on human aging and cancer at the University of Pennsylvania; and Mala Murthy, a prominent neuroscientist now at Princeton.

Again and again I have been greatly privileged in the matter of those who work around me. And that was never truer than it was in Guarente’s lab at MIT. It was a dream team, and I often felt humbled by the people with whom I was surrounded.

When I began my career in this field, I dreamt of publishing just one study in a top-tier journal. During those years, our group was publishing one every few months.

We demonstrated that the redistribution of Sir2 to the nucleolus is a response to numerous DNA breakages, which happen as a result of ERCs multiplying and inserting back into the genome or joining together to form superlarge ERCs. When Sir2 moves to combat DNA instability, it causes sterility in old, bloated yeast cells. That was the first step of the survival circuit, though at the time we had no idea that it was as ancient and as essential to our very existence as it turned out to be.

We told the world that we could give yeast a Werner-like syndrome, causing exploded nucleoli.[62 - D. A. Sinclair, K. Mills, and L. Guarente, “Accelerated Aging and Nucleolar Fragmentation in Yeast SGS1 Mutants,” Science 277, no. 5330 (August 29, 1997): 1313–16, https://www.ncbi.nlm.nih.gov/pubmed/9271578.] We described the ways in which mutants of SGS1, those we’d plagued with the yeast equivalent to the Werner syndrome mutation, accumulated ERCs more rapidly, leading to premature aging and a shortened lifespan.[63 - Sinclair and Guarente, “Extrachromosomal rDNA Circles—A Cause of Aging in Yeast.”] Crucially, by demonstrating that if you add an ERC to young cells they age prematurely, we had crucial evidence that ERCs don’t just happen during aging, they cause it. And by artificially breaking the DNA in the cell and watching the cellular response, we showed why sirtuins move—to help with DNA repair.[64 - K. D. Mills, D. A. Sinclair, and L. Guarente, “MEC1-Dependent Redistribution of the Sir3 Silencing Protein from Telomeres to DNA Double-Strand Breaks,” Cell 97, no. 5 (May 28, 1999): 609–20, https://www.ncbi.nlm.nih.gov/pubmed/10367890.] That turned out to be the second step of the survival circuit.[65 - Sinclair, Mills, and Guarente, “Accelerated Aging and Nucleolar Fragmentation in Yeast SGS1 Mutants.”] The DNA damage that gave rise to the ERCs was distracting Sir2 from the mating-type genes, causing them to become sterile, a hallmark of yeast aging.

It was epigenomic noise in its purest form.

It took another twenty years to learn if those findings in yeast were relevant to organisms more complex than yeast. We mammals have seven sirtuin genes that have evolved a variety of functions beyond what simple SIR2 can do. Three of them, SIRT1, SIRT6, and SIRT7, are critical to the control of the epigenome and DNA repair. The others, SIRT3, SIRT4, and SIRT5, reside in mitochondria, where they control energy metabolism, while SIRT2 buzzes around the cytoplasm, where it controls cell division and healthy egg production.

There had been many clues along the way. Brown University’s Stephen Helfand showed that adding extra copies of the dSir2 gene to fruit flies suppresses epigenetic noise and extends their lifespan. We found that SIRT1 in mammals moves from silent genes to help repair broken DNA in mouse and human cells.[66 - P. Oberdoerffer, S. Michan, M. McVay, et al., “SIRT1 Redistribution on Chromatin Promotes Genomic Stability but Alters Gene Expression During Aging,” Cell 135, no. 5 (November 28, 2008): 907–18, https://www.cell.com/cell/fulltext/S0092-8674(08)01317-2; Z. Mao, C. Hine, X. Tian, et al., “SIRT6 Promotes DNA Repair Under Stress by Activating PARP1,” Science 332, no. 6036 (June 2011): 1443–46, https://www.ncbi.nlm.nih.gov/pubmed/21680843.] But the true extent to which the survival circuit is conserved between yeast and humans wasn’t fully known until 2017, when Eva Bober’s team at the Max Planck Institute for Heart and Lung Research in Bad Nauheim, Germany, reported that sirtuins stabilize human rDNA.[67 - A. Ianni, S. Hoelper, M. Krueger, et al., “Sirt7 Stabilizes rDNA Heterochromatin Through Recruitment of DNMT1 and Sirt1,” Biochemical and Biophysical Research Communications 492, no. 3 (October 21, 2017): 434–40, https://www.ncbi.nlm.nih.gov/m/pubmed/28842251/.] Then, in 2018, Katrin Chua at Stanford University found that, by stabilizing human rDNA, sirtuins prevent cellular senescence—essentially the same antiaging function as we had found for sirtuins in yeast twenty years earlier.[68 - The authors show how SIRT7, in protecting against the instability of rDNA, also guards against the death of human cells. S. Paredes, M. Angulo-Ibanez, L. Tasselli, et al., “The Epigenetic Regulator SIRT7 Guards Against Mammalian Cellular Senescence Induced by Ribosomal DNA Instability,” Journal of Biological Chemistry 293 (July 13, 2018): 11242–50, http://www.jbc.org/content/293/28/11242.]

That was an astonishing revelation: over a billion years of separation between yeast and us, and, in essence, the circuit hadn’t changed.

By the time those findings appeared, though, it was clear to me that epigenomic noise was a likely catalyst of human aging. Two decades of research had already been leading us in that direction.[69 - Oberdoerffer et al., “SIRT1 Redistribution on Chromatin Promotes Genomic Stability but Alters Gene Expression During Aging.”]

In 1999, I moved from MIT across the river to Harvard Medical School, where I set up a new lab on aging. There I was hoping to answer a new question that had increasingly been occupying my thoughts.

I had noticed that yeast cells fed with lower amounts of sugar were not just living longer, but their rDNA was exceptionally compact—significantly delaying the inevitable ERC accumulation, catastrophic numbers of DNA breaks, nucleolar explosion, sterility, and death.

Why was that happening?

THE SURVIVAL CIRCUIT COMES OF AGE

Our DNA is constantly under attack. On average, each of our forty-six chromosomes is broken in some way every time a cell copies its DNA, amounting to more than 2 trillion breaks in our bodies per day. And that’s just the breaks that occur during replication. Others are caused by natural radiation, chemicals in our environment, and the X-rays and CT scans that we’re subjected to.

If we didn’t have a way to repair our DNA, we wouldn’t last long. That’s why, way back in primordium, the ancestors of every living thing on this planet today evolved to sense DNA damage, slow cellular growth, and divert energy to DNA repair until it was fixed—what I call the survival circuit.

Since the yeast work, evidence that yeast aren’t so different from us has continued to accumulate. In 2003, Michael McBurney from the University of Ottawa in Canada discovered that mouse embryos manipulated to be unable to produce one of the seven sirtuin enzymes, SIRT1, couldn’t last past the fourteenth day of development—about two-thirds of the way into a mouse’s gestation period.[70 - M. W. McBurney, X. Yang, K. Jardine, et al., “The Mammalian SIR2alpha Protein Has a Role in Embryogenesis and Gametogenesis,” Molecular and Cellular Biology 23, no. 1 (January 23, 2003): 38–54, https://mcb.asm.org/content/23/1/38.long.] Among the reasons, the team reported in the journal Cancer Cell, was an impaired ability to respond to and repair DNA damage.[71 - R.-H. Wang, K. Sengupta, L. Cuiling, et al., “Impaired DNA Damage Response, Genome Instability, and Tumorigenesis in SIRT1 Mutant Mice,” Cancer Cell 14, no. 4 (October 7, 2008): 312–23, https://www.cell.com/cancer-cell/fulltext/S1535-6108(08)00294-8.] In 2006, Frederick Alt, Katrin Chua, and Raul Mostovslavsky at Harvard showed that mice engineered to lack SIRT6 underwent the typical signs of aging faster along with shortened lifespans.[72 - R. Mostoslavsky, K. F. Chua, D. B. Lombard, et al., “Genomic Instability and Aging-like Phenotype in the Absence of Mammalian SIRT6,” Cell 124 (January 27, 2006): 315–29, https://doi.org/10.1016/j.cell.2005.11.044.] When the scientists knocked out a cell’s ability to create this vital protein, the cell lost its ability to repair double-strand DNA breaks, just as we had showed in yeast back in 1999.

If you are skeptical, and you should be, you might assume these SIRT mutant mice could just be sick and, therefore, short lived. But adding in more copies of the sirtuin genes SIRT1 and SIRT6 does just the opposite: it increases the health and extends the lifespan of mice, just as adding extra copies of the yeast SIR2 gene does in yeast.[73 - The treatments work better in male mice, for reasons that are not yet known, but my former postdoc Haim Cohen at Bar-Ilan University in Israel wins the award for the best-ever name given to a transgenic mouse strain: MOSES. A. Satoh, C. S. Brace, N. Rensing, et al., “Sirt1 Extends Life Span and Delays Aging in Mice Through the Regulation of Nk2 Homeobox 1 in the DMH and LH,” Cell Metabolism 18, no. 3 (September 3, 2013): 416–30, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3794712.] Credit for these discoveries goes to two of my previous colleagues, Shin-ichiro Imai, my former drinking buddy at the Guarente lab, and Haim Cohen, my first postdoc at Harvard.

In yeast, we had shown that DNA breaks cause sirtuins to relocalize away from silent mating-type genes, causing old cells to become sterile. That was a simple system, and we’d figured it out in a few years.

But is the survival circuit causing aging in mammals? What parts of the system survived the billion years, and which are yeast specific? Those questions are on the cutting edge of human knowledge right now, but the answers are beginning to reveal themselves.

What I’m suggesting is that the SIR2 gene in yeast and the SIRT genes in mammals are all descendants of gene B, the original gene silencer in M. superstes.[74 - When we write SIR2 in capitals and italics, it refers to the gene; when we write Sir2, it refers to the protein the gene encodes.] Its original job was to silence a gene that controlled reproduction.

In mammals, the sirtuins have since taken on a variety of new roles, not just as controllers of fertility (which they still are). They remove acetyls from hundreds of proteins in the cell: histones, yes, but also proteins that control cell division, cell survival, DNA repair, inflammation, glucose metabolism, mitochondria, and many other functions.

I’ve come to think of sirtuins as the directors of a multifaceted disaster response corps, sending out a variety of specialized emergency teams to address DNA stability, DNA repair, cell survivability, metabolism, and cell-to-cell communication. In a way, this is like the command center for the thousands of utility workers who descended upon Louisiana and Mississippi in the wake of Hurricane Katrina in 2005. Most of the workers weren’t from the Gulf Coast, but they came, did their level best to fix what was broken, and then went home. Some were working in the storm-ravaged communities for a few days and others for a few weeks before returning to their normal lives. And for most, it wasn’t the first or last time they had done something like that; anytime there’s a mass disaster that impacts utilities, they swoop in to help.

When they’re home, those folks take care of the typical business of being at home: paying bills, mowing lawns, coaching baseball, whatever. But when they’re away, helping keep places like the Gulf Coast from descending into anarchy—a condition that would have had disastrous results for the rest of the nation—a lot of those things have to be put on hold.

When sirtuins shift from their typical priorities to engage in DNA repair, their epigenetic function at home ends for a bit. Then, when the damage is fixed and they head back to home base, they get back to doing what they usually do: controlling genes and making sure the cell retains its identity and optimal function.

But what happens when there’s one emergency after another to tend to? Hurricane after hurricane? Earthquake after earthquake? The repair crews are away from home a lot. The work they normally do piles up. The bills come due, then overdue, and then the folks from collections start calling. The grass grows too long, and soon the president of the neighborhood association is sending nastygrams. The baseball team goes coachless, and the team devolves into the Bad News Bears. And most of all, one of the most important things they do while at home—reproducing—doesn’t get done. This form of hormesis, the original survival circuit, works fine to keep organisms alive in the short term. But unlike longevity molecules that simply mimic hormesis by tweaking sirtuins, mTOR, or AMPK, sending out the troops on fake emergencies, these real emergencies create life-threatening damage.

What could cause so many emergencies? DNA damage. And what causes that? Well, over time, life does. Malign chemicals. Radiation. Even normal DNA copying. These are the things that we’ve come to believe are the causes of aging, but there is a subtle but vital shift we have to make in that manner of thinking. It’s not so much that the sirtuins are overwhelmed, though they probably are when you are sunburned or get an X-ray; what’s happening every day is that the sirtuins and their coworkers that control the epigenome don’t always find their way back to their original gene stations after they are called away. It’s as if a few emergency workers who came to address the damage done in the Gulf Coast by Katrina had lost their home address. Then disaster strikes again and again, and they must redeploy.

Wherever epigenetic factors leave the genome to address damage, genes that should be off, switch on and vice versa. Wherever they stop on the genome, they do the same, altering the epigenome in ways that were never intended when we were born.

Cells lose their identity and malfunction. Chaos ensues. The chaos materializes as aging. This is the epigenetic noise that is at the heart of our unified theory.

How does the SIR2 gene actually turn off genes? SIR2 codes for a specialized protein called a histone deacetylase, or HDAC, that enzymatically cleaves the acetyl chemical tags from histones, which, as you’ll recall, causes the DNA to bundle up, preventing it from being transcribed into RNA.

When the Sir2 enzyme is sitting on the mating-type genes, they remain silent and the cell continues to mate and reproduce. But when a DNA break occurs, Sir2 is recruited to the break to remove the acetyl tags from the histones at the DNA break. This bundles up the histones to prevent the frayed DNA from being chewed back and to help recruit other repair proteins. Once the DNA repair is complete, most of the Sir2 protein goes back to the mating-type genes to silence them and restore fertility. That is, unless there is another emergency, such as the massive genome instability that occurs when ERCs accumulate in the nucleoli of old yeast cells.

For the survival circuit to work and for it to cause aging, Sir2 and other epigenetic regulators must occur in “limiting amounts.” In other words, the cell doesn’t make enough Sir2 protein to simultaneously silence the mating-type genes and repair broken DNA; it has to shuttle Sir2 between the various places on an “as-needed” basis. This is why adding an extra copy of the SIR2 gene extends lifespan and delays infertility: cells have enough Sir2 to repair DNA breaks and enough Sir2 to silence the mating-type genes.[75 - It’s possible that by not allowing mating-type genes to turn on, yeast with additional copies of SIR2 have less efficient DNA repair by homologous recombination, which is what the expression of mating-type genes also does when switched on besides preventing mating. This needs to be tested. But at least under safe lab conditions, the cells grow perfectly fine.]

Over the past billion years, presumably millions of yeast cells have spontaneously mutated to make more Sir2, but they died out because they had no advantage over other yeast cells. Living for 28 divisions was no advantage over those that lived for 24 and, because Sir2 uses up energy, having more of the protein may have even been a disadvantage. In the lab, however, we don’t notice any disadvantage because the yeast are given more sugar than they could possibly ever eat. By adding extra copies of the SIR2 gene, we gave the yeast cells what evolution failed to provide.

If the information theory is correct—that aging is caused by overworked epigenetic signalers responding to cellular insult and damage—it doesn’t so much matter where the damage occurs. What matters is that it is being damaged and that sirtuins are rushing all over the place to address that damage, leaving their typical responsibilities and sometimes returning to other places along the genome where they are silencing genes that aren’t supposed to be silenced. This is the cellular equivalent of distracting the cellular pianist.

To prove that, we needed to break some mouse DNA.

It’s not hard to intentionally break DNA. You can do it with mechanical shearing. You can do it with chemotherapy. You can do it with X-rays.

But we needed to do it with precision—in a way that wouldn’t create mutations or impact regions that affect any cellular function. In essence, we needed to attack the wastelands of the genome. To do that, we got our hands on a gene similar to Cas9, the CRISPR gene-editing tool from bacteria that cuts DNA at precise places.

The enzyme we chose for our experiments comes from a goopy yellow slime mold called Physarum polycephalum, which literally means “many-headed slime.” Most scientists believe that this gene, called I-PpoI, is a parasite that serves only to copy itself. When it cuts the slime mold genome, another copy of I-PpoI is inserted. It is the epitome of a selfish gene.

That’s in a slime mold, its native habitat. But when I-PpoI finds itself in a mouse cell, it doesn’t have all the slime mold machinery to copy itself. So it floats around and cuts DNA at just a few places in the mouse genome, and there is no copying process. Instead, the cell has no problem pasting the DNA strands back together, leaving no mutations, which is exactly what we were looking for to engage the survival circuit and distract the sirtuins. DNA-editing genes such as Cas9 and I-PpoI are nature’s gifts to science.

To create a mouse to test the information theory, we inserted I-PpoI into a circular DNA molecule called a plasmid, along with all the regulatory DNA elements needed to control the gene, and then inserted that DNA into the genome of a mouse embryonic stem cell line we were culturing in plastic dishes in the lab. We then injected the genetically modified stem cells into a 90-cell mouse embryo called a blastocyst, implanted it into a female mouse’s uterus, and waited about twenty days for a baby mouse to show up.

This all sounds complicated, but it’s not. After some training, a college student can do it. It’s such a commodity these days, you can even order a mouse out of a catalog or pay a company to make you one to your specifications.

The baby mice were born perfectly normal, as expected, since the cutting enzyme was switched off at that stage. We called them affectionately “ICE mice,” ICE standing for “Inducible Changes to the Epigenome.” The “inducible” part of the acronym is vital—because there’s nothing different about these mice until we feed them a low dose of tamoxifen. This is an estrogen blocker that is normally used to treat human cancers, but in this case, we’d engineered the mouse so that tamoxifen would turn on the I-PpoI gene. The enzyme would go to work, cutting the genome and slightly overwhelming the survival circuit, without killing any cells. And since tamoxifen has a half-life of only a couple of days, removing it from the mice’s food would turn off the cutting.

The mice might have died. They might have grown tumors. Or they might have been perfectly fine, no worse off than if they’d received a dental X-ray. Nobody had ever done this before in a mouse, so we didn’t know. But if our hypothesis about epigenetic instability and aging was correct, the tamoxifen would work like the potion that Fred and George Weasley used to age themselves in Harry Potter and the Goblet of Fire.

And it worked. Like wizardry, it did.

During the treatment, the mice were fine, oblivious to the DNA cutting and sirtuin distraction. But a few months later, I got a call from a postdoc who was taking care of our lab’s animals while I was on a trip to my lab in Australia.

“One of the mice is really sick,” she said. “I think we need to put it down.”