SENSible Question: Wouldn’t Worms Be Faster?

A supporter asks us if it would accelerate research progress much faster if did most testing in far shorter-lived animals, like the roundworm C. elegans or the fruit fly Drosophila - rather than mice.

SENSible Question: One of the reasons scientists test longevity therapeutics in mice first instead of people is that lifespan studies would be impractically long in humans, whereas they can be done in 3-4 years in mice. Wouldn’t it actually accelerate progress much faster if we instead did most testing in far shorter-lived animals, like the roundworm C. elegans or the fruit fly Drosophila?

On its face, that’s a totally reasonable question: time is ticking for all of us, and we want to get longevity therapeutics into people’s hands as quickly as possible! And certainly these short-lived animals have taught us a lot about the roles of different biological signaling pathways (such as the mTOR pathway or the insulin/IGF-1 pathway) in causing and controlling the biochemical accidents that become cellular and molecular aging damage. Some of those discoveries have in turn proved to also work in mammals, such as mutations in the two pathways we’ve just mentioned, although the insulin/IGF-1 pathway is more complicated in mammals, and inhibiting it leads to much smaller anti-aging effects. And some of that is because these organisms’ life cycles are so drastically different from our own. For instance, a recent study found that knocking out the gene for C. elegans version of an insulin/IGF-1 receptor has a life-extending effect that has no equivalent in mice, (wo)men, or any other mammal: different mutations in this pathway affect different causes of death, and one of them keeps the worms alive by preventing their eggs from hatching inside of them and killing them!
A very different case of death by offspring. Screen capture from Alien; credit 20th Century Fox.

Additionally, some interventions that work in C. elegans act by altering the worms’ early developmental processes, which isn’t terribly helpful to those of us who (as founding CSO de Grey is fond of saying) “have the misfortune of already being alive.” That’s also becoming increasingly evident in mice. We’ve known for about twenty years now that mutations that block IGF-1 signaling in mice slow down their aging and extend lifespan. But those mutations dampen down signaling through these pathways throughout the animals’ entire lives, from the time they’re in the womb until the day they die. To take advantage of that discovery and develop a longevity therapeutic that would work in middle-aged and older adults, a large part of the anti-aging effect would have to be due to the hormone still being low during adulthood.

Instead, studies have shown that almost all the benefit of the “Ames mutation” goes away if growth hormone production is brought back to normal during the very earliest period of life — before the mouse has even stopped suckling its mother, let alone developed into a mature adult. After that, it’s too late to benefit from low growth hormone levels: female Ames mice live no longer than normal mice, and male mice only a little longer, despite being in in a low-growth hormone state throughout adolescence and adulthood.

Almost all the anti-aging benefit of a mutation that prevents growth hormone production in mice is explained by the hormone’s absence during infancy. If scientists restore the growth hormone levels of Ames dwarf mice to normal during this narrow developmental window, the fact that they live their adolescence and adulthood in a low-growth hormone state doesn’t benefit them. Credit: FASEB J 24(12):5073-9 (left), Elife 6:e24059 (right).
And on the other hand, when scientists treat mice that have already reached the mouse equivalent of 60 years of (human) aging with monoclonal antibodies that block IGF-1 from transmitting its signal to cells, it fails to increase lifespan at all in males. In fact, it’s worse than that: surprisingly, the animals instead die with an even heavier burden of tumors at death than untreated animals do. The same antibodies do initially reduce deaths in older females — but without moving the needle on maximum longevity, and at the cost of worsening their age-related muscle loss. And some mutations that extend lifespan in these animals are not just ineffective in humans: they’re pathological. For example, mutations in the genes for some mitochondrial components increase the production of mitochondrial free radicals and lengthen the lives of C. elegans. But in humans, those same mutations instead cause terrible mitochondrial congenital diseases such as Leigh syndromeFriedreich’s AtaxiaSengers syndrome, and progressive external ophthalmoplegia. Because some metabolic pathways play similar roles in driving aging damage across the animal kingdom, one might expect that some of the traditional “messing-with-metabolism”-type longevity therapeutics that work in these simpler organisms would also work in mammals like mice (and us). That’s true in some cases (the standout example in laboratory rodents is rapamycin), but the track record shows that drugs that slow aging in C. elegans and Drosophila often don’t work out in mice. In a recent study, researchers went through lifespan studies that had shown lifespan-extending effects of particular drugs and supplements in roundworms and flies and then looked to see if the same molecules had also lengthened life in mice. First, they looked at all such studies that had ever been curated in our colleague João Pedro de Magalhães’ DrugAge database. They identified 37 drugs and supplements that had been tested in one, the other, or both of the lower species, and had also been tested in mice. But only 56% of agents that had worked in the worms wound up working in mice. The chances were better in Drosophila, though still not a slam dunk: about two-thirds were successful. But the bar in this initial analysis was pretty low for all three organisms because the researchers included all the studies they could find without regard to their quality or reproducibility. And as we’ve discussed before, there are a lot of garbage lifespan studies in the published scientific literature. So they followed up with a second analysis where they only considered C. elegans and mouse studies that were part of two structurally high-quality lifespan study programs for these organisms: the National Institute on Aging (NIA) ’s Interventions Testing Program (ITP) in mice and the Caenorhabditis Interventions Testing Program (CITP) in worms. We’ve talked about the ITP program before: see the section “Failing the Gold Standard” in a previous post about its test of the diabetes drug metformin for lifespan. It’s an effort to test different drug-type longevity therapeutics using a systematic design that ensures that the results are as reliable as possible from the very first report. The CITP is an attempt to apply the same rigorous design to lifespan studies in the roundworm, with the additional advantage that — since testing in C. elegans is so cheap — they can effectively run multiple separate “colonies” of worms through the same study in each of the three independent labs. Since there is no “ITP for flies,” the researchers carefully combed through the Drosophila lifespan studies to make sure they met quality benchmarks. Then they made apples-to-apples comparisons between the results of these more rigorous fly lifespan studies with worm and mouse studies conducted with the same drugs and supplements by the CITP and ITP (respectively). But the results came back more or less the same as the first analysis: only half of the drugs and supplements that extended the lives of CITP worms did so in mice, and only three-quarters of those that passed the bar in quality Drosophila studies worked out in ITP mouse studies. And that probably overstates how well a study first conducted in C. elegans can predict success in mice, because many of the drugs that the CITP tested were included for testing precisely because they had already worked for mice in the ITP. So it’s a mistake to count such studies as evidence that testing longevity therapeutics in short-lived worms first would save time by highlighting promising candidates for mice when it actually happened the other way around. By contrast, one of the key reasons to favor “damage-repair” strategies is that they undo existing aging damage and don’t depend on manipulating developmental or any other metabolic process. Conceptually, that gives SENS rejuvenation biotechnology much more power than conventional “messing-with-metabolism” longevity therapeutics to postpone age-related illness and death when your first chance to take advantage of them happens when you’re already in or past what is currently “middle age.” (That’s the same reason why effectiveness later in life is a key principle of the Robust Mouse Rejuvenation (RMR) goalpost). Plus, some of the worm results were worse than just not translatable to mice. For instance, N-acetylcysteine (NAC) and glutathione accelerate aging in C. elegans, but it either has no effect on (females) or slightly increases life expectancy in (male) mice. (Before you rush down to the supplement aisle, note that there’s a bit of a wrench in this result: about two weeks into the experiment, the mice fed NAC-fortified chow suddenly started losing weight — and while the females stabilized at a new, lower weight, the males just kept dropping grams. You might think that a combination life extension and weight loss drug is like getting some chocolate with your peanut butter. But actually, the weight loss suggests that NAC may have caused the animals to eat less, in which case the apparent life extension in males might simply be the result of unintended Calorie restriction. That would be consistent with the fact that NAC only “worked” (if it did) in males. Whatever the case, we can say at least that NAC did not put the mice’s aging process on fast-forward as it did for the worms).
NAC may have a very small longevity benefit in male mice, though not in females. But might it be due to the supplement causing them to lose their appetites? Credit: J Gerontol A Biol Sci Med Sci 65(12):1275-84.

Crawling Our Way Forward?

But all of the preceding examples apply to studies based on trying to usurp the regulation of metabolism to slow the aging process down. SENS Research Foundation is instead grounded in the direct “damage-repair” strategy of SENS. If we’re going to use an organism as a test animal for rejuvenation biotechnology, it has to accumulate similar kinds of aging damage as we do, and it must do so in similar tissues and with similar pathological results. And here C. elegans and Drosophila just aren’t qualified for the job.

For instance, C. elegans don’t live long enough to accumulate cells overtaken by mitochondrial DNA deletions, and there is no clear link between other kinds of mitochondrial DNA damage and the rate of aging in these worms. It is not clear to this author whether nuclear DNA accumulations accumulating in the cells of aging C. elegans contribute to their aging either — but if they do, it’s not through causing cancer, since the worms don’t develop this disease because their mature bodies are composed entirely of cells that don’t divide. Granted that the medium-term “damage-repair” strategy for nuclear mutations is first and foremost to build an impregnable wall against cancer, the fact that they don’t develop cancer again makes them a lousy model for testing SENS therapies (especially when shooting for the kind of multi-component approach that’s needed to reach Robust Mouse Rejuvenation (RMR)).

The lack of dividing cells is also why C. elegans aren’t susceptible to the many diseases that afflict epithelial tissues like the skin or the linings of many of our organs. That includes atherosclerotic cardiovascular disease (ASCVD), the disease of the arteries that is the number one single-disease cause of death worldwide. In fact, C. elegans are so small that they don’t even have hearts or circulatory systems as we would recognize them in mammals. Instead, the worms rely on diffusion and the rhythmic pumping of their feeding tube to spread oxygen, waste gas, and fuels around their tiny bodies. And the major arteries are possibly one of the first places in the body where rejuvenation biotechnology will target AGE crosslinking, another target for SENS. So already, that’s the two greatest causes of death in humans that are off the table if C. elegans are the test animal of choice.

C. elegans also have no bones, so no osteoarthritis or osteoporosis either. And they lack any of the cells dedicated to the immune system. Instead, one of their primary defenses against pathogens is called “behavioral avoidance” — meaning that they literally run (all right: wriggle) away from dangerous microbes! So the worm model is also blind to the catastrophic failure of the immune system with age, and we can’t meaningfully use them to test damage-repair strategies to remedy it.

With all those causes of death passing by C. elegans, what exactly ends their incredibly short lives? Mostly, it appears to be pathologies of their feeding tube.

Fly Away Home?

Although significantly closer to mammals than roundworms are, the fruit fly Drosophila also ages very differently from mammals like us at the structural level. For instance, Drosophila don’t lose dopaminergic neurons in their brains as they age. These specialized neurons produce the signaling molecule dopamine, and their loss with age is responsible for the more prominent symptoms of Parkinson’s disease. Their brains also don’t naturally suffer the age-related accumulation of the most important intracellular and extracellular aggregates that drive this and other neurodegenerative aging diseases, unless scientists genetically modify them with mutated versions of these human proteins.

(It’s often said that mice and rats don’t either — but this isn’t quite true. Completely normal Fisher 344 x Brown Norway rats accumulate soluble beta-amyloid protein as they age, albeit not plaque, and so do mice subjected to experimental high blood pressure. And normal Wistar-Kyoto rats develop neurofibrillary tangles, though the tangles are restricted to the branching arms of the neuron instead of extending into the cell’s main body. Plus, both mice and rats accumulate lipofuscin in their brains as they age — lipofuscin that is chemically very similar to the junk in human brains. Instead, shortly before a Drosophila dies, a mysterious sudden spike of an unknown fluorescent compound occurs in their eyes and elsewhere in their bodies (but not specifically in their brains), leaving a scene straight out of a 1950s zombie movie.

An unknown fluorescent material suddenly appears in the eyes and bodies of Drosophila just before they die. Credit: Exp Gerontol 126: 110707.

Drosophila have been useful for studying the genetic and biochemical basis of excess cell proliferation and the role of specific genes in cancer biology, but some of the differences are stark. Some of the most important genes that keep our cells from becoming cancerous (such as p53Rb, and APC) don’t even cause cellular overgrowth in the fruit fly, let alone full-on cancer.

And while Drosophila do develop abnormal growths in different tissues, many of these disorders are congenital genetic disorders rather than the result of mutations acquired because of aging. These tumors are not true cancers, and often behave very differently from cancer as it occurs in humans. And the one disorder of excessive cell growth and loss of differentiation that is a prevalent cause of death in aging flies — perhaps the most common — is “the exception that proves the rule:” a growth of abnormal cells kills flies through loss of barrier function in their guts, which is not something we would recognize as cancer.

Whereas C. elegans don’t suffer cancer, and Drosophila tumors only “rhyme” with it, cancer is the number one cause of death in lab mice — and even when they die of cancer, aging mice die with many of the other pathologies of the aging body that plague humans, and accumulate lesions from of all categories of aging damage targeted by SENS.

That said, aging fruit flies’ bodies do share more specific aging pathology with humans than roundworms do. One important example is that — like the human heart — Drosophila heart structure and function also degenerates with age. Their heart muscle cells become increasingly disorganized, they become increasingly susceptible to erratic heartbeats (arrhythmias), and their hearts stiffen due to abnormal extracellular matrix deposition, leading to heart dysfunction that in some ways resembles human heart failure. Their hearts also lose cells as adults — but because of ongoing programmed cell death rather than heart attacks or other insults as happens in humans.

Yet while certainly not good for aging flies, it’s not clear that any of these heart lesions actually cripple or kill them as they so often do in humans. Certainly they don’t get atherosclerosis or hypertension, which are the drivers of cardiovascular death in humans. Indeed, while flies accumulate AGE of a sort, the known AGE species in Drosophila are the molecular equivalent of barnacles rather than the AGE that is the principal target for SENS rejuvenation biotechnology in the major arteries.

By contrast, mice develop human-like heart dysfunctionfibrosisarterial stiffening, and dysfunction of the blood vessels with age. And although they don’t develop atherosclerosis when fed a standard diet, non-mutant mice do develop atherosclerotic lesions if fed a diet overloaded with saturated fat, cholesterol, and “supplemental” bile acids.

Testing longevity therapeutics in Drosophila is also made tricky; complicated by the large differences in lifespan that can result from dietary minutia that would make little difference in the life of a mouse or (wo)man, and by the need to keep the animals free from a parasitic bacterium called Wolbachia that kills most fly strains prematurely. But equally, protocols intended to keep the animals free of this parasite can also shorten the lives of some fly strains. An additional fly in the ointment (the reader will look mercifully on the pun) is that scientists usually deal with Wolbachia by regularly treating the fly colony with antibiotics — but doing so inevitably confounds experiments with the confusing inconsistency of reports of the gut microbiome in fly aging and lifespan, and its likely role in influencing aging and its diseases in humans.

Wolbachia inside the cell of an insect. Credit: Plos Biology, 01 Mar 2004, 2(3). Distributed under Creative Commons Attribution License.

For goodness’ sake, you can substantially extend the lifespan of Drosophila by putting the little pests in the fridge! Even the most enthusiastic fan of Wim Hof can’t expect it to work that way in mammals.

Beyond Biology: Other Reasons to Stick with Mice

We’ve seen above that as a matter of sheer biology, short-lived invertebrates like C. elegans and Drosophila are not good choices to test rejuvenation biotechnologies. But there are other reasons to prefer mice that go beyond such narrowly scientific ones.

For one thing, most people find lab mice to be sympathetic, cute creatures, and their youthful or degenerative aging are obvious to the naked eye. These features make them better animal ambassadors for the effects of rejuvenation biotechnology than creepy-crawlies of one kind or another. Those same characteristics are even more true of our pets, which is one reason why Dr. Matthew Kaeberlein is running “trials” of rapamycin in pet dogs, even though they live even longer than mice do and thus take yet more time to show results. Since part of the goal of RMR is to seize the imagination of the public, this “cuteness factor” gives mice a decided leg up over animals that either don’t have legs at all, or whose legs are supported by an articulated exoskeleton.

Additionally, FDA and its counterparts in other wealthy countries insist that developers test their would-be therapies in mammals, one of which must be a rodent. If the goal is to get our therapies into humans quickly, we save time by cutting out the middle-worm and going directly to mice, which will in turn bring us closer to human trials.

And finally, it’s important to remember that even after completing the mouse and other animal studies required to start human testing, far too many drugs fail to prove themselves safe or effective in humans — many of them at the very first step along the way.

More than 90% of drugs that are safe and effective when tested in experimental animals ultimately fail in human testing. Credit: Redrawn by Anne Corwin from Nat Rev Drug Discov 3(8):711-5.

Because they directly target stable cellular and molecular damage rather than trying to reach them through the tangled pathways of metabolism, “damage-repair” strategies are in principle less prone to foundering on the shoals of the metabolic quirks of some test animal than are conventional drugs. No model is perfect, but we should make the best use of the animal partners we have. All the medicines that have saved human lives today — from antibiotics, to clotbusters, to statins, to recombinant blood proteins for haemophiliacs — went through this path. We can now take it to greater heights by conquering aging, the plague that has been the ultimate target of medicine since the dawn of science.

“from the very moment of your Nativity, you make every day a considerable step toward Old Age, which is itself a Disease … I am persuaded, were men as careful in preserving their Health, as they are solicitous for the recovery of it, they might often multiply the Sum of their Years, and live the Product without a Disease. And I count it a Piece of Skill in a Physician far surpassing the most admirable Cures, to preserve a Man from all Diseases.” - Roger Bacon, The Cure of Old Age and Preservation of Youth. Statue at the Oxford University Museum of Natural History. Photo Credit: Michael Reeve.

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