One of my colleagues is involved with VitaDAO, and based on what I have seen from them it seems like a cool project. Since I have not been involved with any anti-aging communities for a while, I decided to write a bit about one of their upcoming events. As far as I can tell, VitaDAO is like a neat web 3.0 version of longecity, that tries to combine longevity research with crypto, crowd-funding, discord and other techy stuff that is appealing to young people (which is kind of in line with my vision for modern biogerontology). So far I am not sure, however, if the crypto-tech aspect is madness or genius, but only time will tell.
VitaDAO – Crypto meets Longevity Symposium
Date: Wednesday 13th of April starting 4pm CET
Duration: 6-7 hours
The organizers aim to "to educate the Crypto field on Longevity, and the Longevity Biotech sector on Blockchain technology", while I aim to break my eternal writer's block. The biology aspect, for sure, is solid and worth writing about. The symposium will cover conference staples such as senolytics, small molecules, fasting-mimicking diets, and the eternally hot, controversial topic of parabiosis ("young blood"), recently joined by another controversial upstart reprogramming. I will explain why these topics matter and add some of my current thoughts about the state of the art.
Senescence and senolytics
As it has become clear, most species accumulate so-called senescent cells when they grow older. These cells are arrested in a half-dysfunctional state, secreting harmful cytokines and other molecules that promote inflammation or might just generally contribute to entropic noise. This has earned them the endearing name "zombie cells" and removal of such cells in mouse models is one of the more robust healthspan promoting interventions, with some impact on lifespan as well. If you want to delve into this in a bit more detail, quite a bit more detail, in fact, I have written about senescence on this blog before.
Here, I would like to comment on the clinical potential of senolytics and translational pitfalls. It appears that aging researchers and biotech/pharma companies are still trapped in 20th-century-thinking always looking for the "age-related disease most likely to benefit from senolytics". While this kind of study design may be a necessary evil, it is somewhat inconsistent with modern biogerontology. If senescence played a moderate role in dozens of age-related diseases, it would be very difficult to detect changes in disease-specific outcomes in a typical small phase II study. In fact, we need something like the TAME trial or a TAME pilot for senolytics, i.e. a study that focuses on composite outcomes.
The cornerstone of 20th century biogerontology is the finding by Clive McCay that caloric restriction extends the lifespan of white rats (1). In fact, almost every modern intervention (or mutation) that extends lifespan in mice is related to caloric restriction. It is anti-anabolism all the way down, as I have argued. Most anti-aging interventions slow proliferation, growth, maturation or anabolic processes. While this is a useful paradigm, we are definitely trapped and need to escape from doing just this one thing. First of all, we need radically different treatments to significantly delay aging. Aubrey de Grey was never wrong on that account. Second, anti-anabolism carries certain risks, like increasing frailty in the elderly.
The other problem with caloric restriction (CR) is publication and model bias. More and more, we have found out that CR is just not that robust, partly because too often we have studied CR in ugly, unprofessional ways (2). I have discussed the evidence here, and since then I became even more skeptical about the quality of the mouse studies (why? - that shall be left for a future post). Luckily, it seems like rapamycin and other interventions may be a more robust version of CR! It makes sense, as semi-starvation is very stressful for the body and hence older mice, for example, often fail to adapt to CR. It might be better to activate or inhibit the central nutrient-sensing pathways without the actual stress. Interestingly, the human epidemiology (or rather mendelian randomization) recently turned out to support CR somewhat, and when I saw the data I triumphantly titled my post "Biogerontologists Score - Thin is Healthy!"
The other other problem with CR is that no one will do it. Obesity pandemic and all that, you know. Thus people like Valter Longo and others are searching for more palatable approaches. Many ideas have emerged including fasting, time-restricted feeding, moderate protein restriction, single amino acid restriction etc. They all do have varying support from mouse models and epidemiology. I think it is worthwhile research, even though it is never going to make a big dent. Small molecule drugs are a better candidate for anti-aging therapies because compliance is easier.
Small molecules are defined as chemical substances with a mass of below ~900 Da. Why not big molecules you may ask? Small molecules are useful because they have good oral availability, in contrast to biologics and big molecules, are cheaper to produce and easier to use in screening libraries, although I am not sure if the latter is due to an accident of history or a real advantage (5).
Historically, they have been the mainstay of big pharma and have saved millions of lives. By and large, and despite all the controversies, the story of big pharma has been a story of "good capitalism" (3). When the interests of money and of the common people align, great things can happen. If you are interested in the pharma field please read Derek Lowe's blog, he is witty, insightful and sometimes writes about aging research.
For what it is worth, rapamycin has a molecular weight of 914 Da, dasatinib of 488 Da and quercetin of 302 Da (together they form the famous senolytic cocktail D+Q). Clearly, small molecules are of some use to biogerontologists.
Recently one thing on my mind was the limitation of small molecules, as useful as they are. Is there even a hypothetical combination of small molecules that could extend the lifespan of C. elegans to be quasi-immortal given infinite screening power? (4) I do not think so. Of course you may argue it is pointless to think about the far future and quasi-immortality (I disagree, "Let us talk about Immortality"), but the issues run deeper. There may be therapies that are fundamentally more effective than small molecules on a 1-to-1 basis, say, e.g. viral delivery or cell therapies. It is just something to keep in mind and I think parabiosis and "young blood" are such candidates (6).
Parabiosis and "young blood"
A procedure that connects the blood circulation of two different animals is called parabiosis. Why would you want to do that? Parabiosis was invented in the 19th century and later used by 20th-century physiologists to great effect showing, for example, that hormones produced by obese mice could induce starvation in a linked-up control mouse. This hormone turned out to be leptin. It was not until the 2000s when the technique was rediscovered by the group of Thomas Rando at Stanford and his then-student (postdoc?) Irina Conboy, who found that linking up a young organism with an old one might lead to rejuvenation of the old due to circulating factors in young blood. Although this finding still has not lead to any tangible breakthroughs in mice, much less humans, as far as I can tell, it nevertheless made my list of key findings of the last decade (2010-2020). The promise is there and it could become really big.
The most recent development in this field is called neutral blood exchange (Mehdipour et al. 2021). It is based on the idea that, rather than transferring a protective factor from the young to the old, parabiosis simply serves to dilute a pro-inflammatory, pro-aging milieu in the old organism. In this case, blood plasma was removed and replaced with albumin containing solution (a procedure that is well established in the clinic and called apheresis). This study, however, again highlights what I dislike about the parabiosis field specifically and mouse aging research more broadly. A claim is made that healthspan is improved, but only limited, non-standardized evidence is provided. Instead I would like to see the same measure of healthspan used in every mouse study, and on top of that you can do whatever you want. So, please, just use the Whitehead frailty index.
Personally, I also like that parabiosis is conceptually simple, side-stepping many difficult questions, and related to the iron accumulation hypothesis of aging. We know that body iron stores are elevated during aging, especially in middle-aged men, and bloodletting appears to protect from cancer. This is a "dilution" so to say. We know that blood donation might be healthy for that reason (might, because it is still controversial due to healthy user bias). However, I am wondering if anyone looked at the effects of plasma donation on mortality and age-related diseases?
Reprogramming the epigenome
The massive success of the epigenetic clock(s) in predicting chronologic age hinted at the possibilities here. Something clearly changes in the epigenome with age, although figuring out causality was never going to be easy. David Sinclair was one of the first researchers who wanted to test this experimentally in animals using so-called ICE mice, which are supposed to harbor non-mutagenic DNA breaks that disturb local epigenetic marks (Hayano et al. 2019), but as much of his work this also remains controversial (8).
There are two types of reprogramming, both useful for the aging researcher. First came the reprogramming of somatic cells into induced pluripotent stem cells. A technique that has its use both as study tool, because it greatly improves in vitro models of disease and, eventually, might help to produce cells, tissues and organs for replacement therapies. In 2012 this culminated with a Nobel prize for Gurdon and Yamanaka. Later on, and that is what we are talking about here, scientists figured that if epigenetic damage accumulated with aging, reprogramming, or what they term partial reprogramming, might be able to revert this in a living organism.
Recently some studies started emerging to support this idea. It does not matter if ICE mice are a good progeroid model showing premature aging, if you can actually rejuvenate mice with partial reprogramming. Most of the data was done in cell culture (and not without its own controversy) and there is also some promising data from a progeroid model (Ocampo et al. 2016, also magically attracting criticism). However, on this blog, we prefer to look at healthy, well-husbanded mice as the gold standard.
The Altos Labs publication by Browder et al. 2022 was supposed to do that. It received quite a lot of attention on science twitter and it is one of the best studies the field has produced so far (for better or worse). From the beginning, when you see supposedly groundbreaking data, you can ask yourself why was this not published in Nature or Science if it is so great? (7) At any rate, this one was published in Nature Aging which is also among the best aging journals in the world.
There are two ways of reading a paper. One is to have a quick glance and the other is to look at it in varying levels of detail. As researchers we have to take these shortcuts because we cannot read every paper that comes out in detail or else we would spend 26 hours per day just assessing literature. When this publication first came out, I had a quick look and immediately noticed a major weakness. Except for figure 5, it is all biomarker work (epigenetics, transcriptomics, metabolomics and lipidomics), so we have no clue whether it works or not! The next question is if the biomarker work is any good? Let us have a look.
The authors tested two long-term reprogramming strategies, induction from 15 to 22 months and from 12 to 22 months. Reprogramming is achieved through cyclical expression of the classic reprogramming transcription factors Oct3/4, Sox2, Klf4, c-Myc (abbreviated with OSKM) under a doxycycline-inducible promoter. Expression is induced for 2 days followed by a 5 day break.
No difference in survival was seen, which is reported as a positive outcome when it is not, although to be fair their study was underpowered to detect any differences.
Next, the authors show epigenetic rejuvenation using “Lifespan Uber Correlation (LUC) clocks, which are constructed on the basis of CpGs whose rate of change correlates with maximum life span across mammals” in 2 out of 6 tissues, whereas a more conventional clock only shows rejuvenation in skin (and several of the p-values would barely pass a Bonferroni correction for multiple testing). A priori there is no reason to think that an endogenous signaling pathway would preferentially induce changes in methylation of CpG islands that have been selected by evolution across species rather than those that more strongly change within species. Clearly, the LUC clocks are not appropriate for this task unless the authors can show within species validation (10). They are not invalid, but as of now, we have to assume they are inferior to conventional clocks.
It stands to reason that the LUC clock was chosen to improve the look of the manuscript. Indeed using standard elastic net (EN) clocks we can only see rejuvenation in the skin after long-term reprogramming and during short-term reprogramming the kidney cannot decide whether it is older or younger.
Fig S6 from Browder et al: note the kidney is misbehaving againAfterwards the authors focus a lot on the skin, because if I am honest, it looks like the treatment only really works in skin. This makes sense since these protocols were largely developed and validated in vitro on fibroblasts.
The RNAseq data is also not very convincing. The authors state that “In the skin, for example, we observed a clear separation of skin samples from control and doxycycline-treated mice”, but that is not immediately obvious from the shown PCA plot, perhaps, because the sample size is at the very low end of what is acceptable for this type of analysis. Also a bit frustrating is the reliance on PCA plots and differential expression analysis of hand-selected pathways to begin with. Is there nothing more elegant like a mouse transcriptomic aging clock or signature? I am aware of transcriptomic clock work in humans or C. elegans, but at a quick glance I could not find anything (published) in mice, although I do recall that Vera Gorbunova presented a clock derived from cross-species data (9).
The other recent publication that made a splash in the field was Lu et al. (2020) from the Sinclair lab, who showed that reprogramming with OSK, leaving out the oncogenic c-Myc, could restore mouse vision at 12 but not at 18 months of age. Without going into much detail, they showed that reprogramming reversed the age-related transcriptomic signature seen in the eye (retinal ganglion cells), which to me seems like the data that Browder et al. should have shown.
I like crazy ideas, however, reprogramming still needs some more work. Rigorous replication and validation is key so the paradigm does not get stuck with mediocre healthspan and lifespan studies like telomerase research did for years. Again I am frustrated that the authors fail to discuss the shortcoming of their paper, instead we have to rely on pubpeer and twitter for critical discussion.
References and notes
epistemic status: relatively quick write-up and brainstorming, my expertise is in mouse longevity
1/ McDonald, Roger B., and Jon J. Ramsey. "Honoring Clive McCay and 75 years of calorie restriction research." The Journal of nutrition 140.7 (2010): 1205-1210.
2/ have you ever seen the funnel plots of CR mouse studies? Or the reporting quality and sample sizes of a typical mouse aging or CR study? Sadly, it is pitiful.
4/ let's define quasi-immortality as a lifespan of 10 years without abusing any of the Dauer stage pathways and no massive loss in fecundity or vitality.
5/ I do not know how one would go about screening biologicals, considering that many of these require a systemic response.
6/ 1-to-1 basis means small molecule vs other intervention in terms of treatment cost, developmental cost or efficacy. It may well be that for every billion dollars spent on small molecules, one could achieve more if the same money was spent on non-conventional therapies.
7/ one obvious alternative explanation is that failing to publish in the top-tier journals has nothing to do with the manuscript quality, Nature and Science suck, they tend to publish overhyped papers and miss a lot of outstanding work that ends up in Elife, Cell Metabolism or Aging Cell.
8/ It was argued that he failed to convincingly demonstrate lack of mutagenic DNA damage in his model.
9/ I was not very impressed with the validation given that MR and CR are life-extending but score very low.
10/ The LUC clocks were never developed with the goal of being the superior clock. As stated in the original Horvath publication:
"We used LUC-related CpGs to develop novel epigenetic age estimators for mice. These LUC clocks do not outperform existing mouse clocks in terms of accuracy of chronological age. Rather, these clocks are meant to address a more elusive aim: the measurement of biological age that correlates with lifespan. The LUC clocks are based on the premise that LUC-related CpGs, being also linked to lifespan, are more informative of biological age than purely age-related CpGs [even this is a stretch because intraspecies life extension mechanisms and interspecies life extension mechanisms are often quite different]. However, this hypothesis requires validation."
Keep in mind, the authors did indeed provide minimal validation using GHRKO and CR mice, showing that these are younger with the LUC-based clock. Unfortunately, there is no validation on skin and kidney that I am aware of.
Mehdipour, Melod, et al. "Plasma dilution improves cognition and attenuates neuroinflammation in old mice." GeroScience 43.1 (2021): 1-18.
Whitehead, Jocelyne C., et al. "A clinical frailty index in aging mice: comparisons with frailty index data in humans." Journals of Gerontology Series A: Biomedical Sciences and Medical Sciences 69.6 (2014): 621-632.
DNA Break-Induced Epigenetic Drift as a Cause of Mammalian Aging
doi: 10.1101/808659 doi: 10.2903/j.efsa.2012.2904 issn: 1831-4732
Hayano et al.
Ocampo, Alejandro, et al. "In vivo amelioration of age-associated hallmarks by partial reprogramming." Cell 167.7 (2016): 1719-1733.
Browder, Kristen C., et al. "In vivo partial reprogramming alters age-associated molecular changes during physiological aging in mice." Nature Aging 2.3 (2022): 243-253.
Lu, Yuancheng, et al. "Reprogramming to recover youthful epigenetic information and restore vision." Nature 588.7836 (2020): 124-129.
Senescence: updated evidence. March 25, 2020.
CR Plus, CR adjacent, CR independent - what the heck? September 04, 2020
Caloric Restriction and Fasting - does it work? December 21, 2021
Biogerontologists Score - Thin is Healthy! August 29, 2021
Let us talk about Immortality. October 12, 2021
Roughly a decade of progress in biogerontology. January 03, 2021.
In defense of being underweight. August 23, 2017.
Telomeres vs DNA damage - battle of the aging theories. March 26, 2021.