Lifespan extended 500 percent

Researchers at the Buck Institute altered two genes in a roundworm and watched it live five times longer, an effect larger than either gene gave alone.

A worm that lived twice as long

In December 1993, Cynthia Kenyon and colleagues at the University of California, San Francisco reported in Nature that mutations in a single gene, daf-2, allowed adult Caenorhabditis elegans hermaphrodites to live more than twice as long as wild type while remaining fertile and active. At the time it was the largest lifespan extension recorded in any organism. The extension required a second gene, daf-16. Both genes were already known to control entry into the dauer, a long-lived arrested larval form that the worm adopts under crowding and starvation. Kenyon's reading was that the longevity of the dauer was not a side effect of arrested growth but a regulated programme, and that daf-2 and daf-16 were the entry points to it. daf-2 was later shown to encode the worm's insulin and IGF-1 receptor; daf-16 encodes the transcription factor FOXO.

The second pathway

A second lifespan axis emerged over the following decade around the nutrient sensor TOR and the control of protein synthesis. In 2007, two papers in Aging Cell published back to back reached the same conclusion. Malene Hansen and Kenyon at UCSF found that reducing ribosomal proteins, translation initiation factors or the ribosomal protein S6 kinase extended lifespan and increased heat-stress resistance, and that some of these effects ran through DAF-16 while others did not. Kah Zhen Pan, Pankaj Kapahi and colleagues at the Buck Institute for Age Research found that inhibiting ifg-1, the worm's eIF4G, or rsks-1, the worm's S6 kinase, extended lifespan while slowing development and reducing fecundity. The 2013 paper would later cite the rsks-1 effect from that work as a 24 percent extension.

Two pathways, insulin signalling and TOR, each extended life on its own. Both are conserved from worms to mammals. The experiment that followed was to disable both in the same animal and measure whether the effects added, interfered, or did something else.

Two mutations, one animal

Di Chen, then a postdoctoral fellow in Kapahi's laboratory at the Buck Institute and by publication an associate professor at Nanjing University, built the double mutant. It carried daf-2(e1370), a strong loss-of-function allele of the insulin receptor, and rsks-1(ok1255), a deletion of the S6 kinase gene. Animals were scored as alive, dead or lost every two to three days, and the wild-type N2 control was run alongside the mutants in at least six independent experiments. Kapahi later said, in a Buck Institute account of the work, that the number of lifespan experiments Chen ran was remarkable.

The paper, published in Cell Reports on December 26, 2013, reports the numbers plainly. The rsks-1 single mutant increased mean lifespan by 20 percent. The daf-2 single mutant increased it by 169 percent. The double mutant increased it by 454 percent relative to wild type, which the authors describe as a nearly five-fold lifespan extension. Had the two effects been additive, the expected combined extension would have been near 189 percent. The observed figure was more than twice that. The paper's title gives the word the authors chose for it: synergistic.

Where the synergy came from

The rest of the paper is an attempt to locate the multiplication. Three findings carry it. First, the synergy required the energy sensor AMPK. In the presence of its catalytic subunit aak-2, deleting rsks-1 extended the mean lifespan of the daf-2 mutant by 85 percent; without aak-2, the same deletion added only 18 percent, an additive effect. The authors describe a positive feedback loop in which AMPK amplifies the activity of DAF-16, the FOXO factor that Kenyon had identified twenty years earlier.

Second, the synergy required a functioning germline. In animals carrying a gain-of-function allele of the germline Notch receptor gene that alters germline signalling, deleting rsks-1 extended daf-2 lifespan by only 14 percent, against 86 percent in animals without it. Third, inhibiting rsks-1 specifically in the germline, using tissue-restricted RNA interference, activated DAF-16 in the intestine, a different tissue. The signal that extended life was being sent from the reproductive cells to the gut. Chen said in the Buck Institute's statement that the germline was the key tissue for the synergistic gain in longevity; Kapahi said the two mutations set off a positive feedback loop in specific tissues that amplified lifespan.

The mechanism, six years later

In July 2019, Jianfeng Lan, Jarod Rollins, Aric Rogers, Kapahi, Chen and colleagues at Nanjing University and the MDI Biological Laboratory returned to the double mutant with polysome profiling, a method that reads which messenger RNAs are being actively translated. They identified translational control of ribosomal genes and of cytochrome c, encoded by cyc-2.1, as mediators of the longevity. Knocking down cyc-2.1 extended lifespan by activating the mitochondrial unfolded protein response, mitochondrial fission and AMPK in the intestine. Again the germline was the key tissue: germline-specific knockdown of cyc-2.1 activated the intestinal stress response at a distance, and the RNA-binding protein GLD-1, which represses cyc-2.1 translation in the germline, was required for the synergistic longevity of daf-2 rsks-1. The 2013 observation had become a pathway with named components.

In human terms

The Buck Institute's statement on December 12, 2013 translated the five-fold figure into a human equivalent of 400 to 500 years, and that comparison has followed the result ever since. It is arithmetic, not biology. The comparison assumes that a worm's lifespan scales to a human's, which no experiment has shown. What does carry across is the architecture. Both pathways exist in humans: the insulin and IGF-1 receptor that daf-2 encodes, and the S6 kinase downstream of TOR that rsks-1 encodes. The paper's closing argument is that interactions between these two conserved pathways, rather than either one alone, deserve attention in more complex organisms.

What the story does and does not show

The 2013 paper showed that combining a strong loss-of-function mutation in the insulin receptor with a deletion of S6 kinase increased the mean lifespan of C. elegans by 454 percent, against 169 percent and 20 percent for the single mutations, in experiments repeated at least six times against wild-type controls. It showed that the excess over additivity depended on AMPK, on DAF-16 and on signalling from the germline to the intestine. The 2019 follow-up gave that signal a molecular route.

The animal is a small nematode with a short natural lifespan, and the measurement is of mean lifespan under laboratory culture. The daf-2(e1370) allele is a strong mutation that also produces developmental and reproductive effects, and the double mutant is not a model of a healthy long-lived animal in any sense that would transfer to a mammal. The 400 to 500 year figure is a proportional conversion offered in a press statement and has no experimental basis. The slide text says one gene alone doubled lifespan; the paper's own figure for daf-2 is 169 percent, which is closer to a tripling. What the result does show, and what changed the field's approach, is that two longevity pathways did not add. They multiplied, and the paper located the tissue in which the multiplication occurred.

Five lifetimes in one animal. Both switches exist in you.