Freitag, 26. Juni 2015

How not to run a lifespan study

Every biogerontologist should have a poster on the wall with the following paper:

Spindler SR. Review of the literature and suggestions for the design of rodent survival studies for the identification of compounds that increase health and life span. Age (Dordr). 2011 Mar 22. [Epub ahead of print]

Why do I care about other people's research so much? One could obviously quip: Why does it matter if people run badly designed lifespan studies? Isn't it up to the principal investigator (PI) to decide if they want to screw with taxpayer and grant money? Well, first of all, I don't think that people run these weak studies on purpose, but I do believe they should know better. It's the PI's job to be on top of current research practise. Admittedly, the mouse facility may be out of your control, but if you cannot guarantee high quality, why would you commit to a 4-year lifespan study? The main issue I have with these weak studies is that they waste more than the money of a single research group or a grant. They also lead to unnecessary follow-up research. Let me give a few examples.

Resveratrol research produced a lot of unwarranted hype that was later defused by the NIA's ITP, yet how many research dollars were spent to do so? The initial study by Sinclair fell into a common trap: methodically weak (2), but not terrible enough to rule it out as a waste of a biogerontologist's time. Due to the hype, it produced a lot of unnecessary follow-up work. Soon afterwards, for reasons completely beyond me, Resveratrol was granted three "slots" in the ITP study at varying doses (a slot here is one whole lifespan study, there are 3-5 slots available per year). Instead, we could have studied something more productive, but hype and perhaps politics got in the way.

Another type of "methodically weak, but not terrible" research plagues the fields of autophagy, glycation and aging. These studies are good enough so there is little grant money to repeat what seems like a redundant study, but weak enough to cast doubt on the results (no replication, crypto-CR,  short-lived controls). To quote myself:
I am not exactly sure what is holding back the field. However, there are 2 promising interventional studies in mice, or perhaps, I should say only two. ATG5 overexpression (7a) in somewhat short-lived strain and hepatic rejuvenation by the Cuervo lab (7b). Unfortunately, Dr. Cuervo has never responded to my inquiries about extending and reproducing her work.
Advanced Glycation Endproducts  
Again, I am not exactly sure what is holding back the field. Since the failure of Alagebrium (around 2013) and some promising studies by Vlassara (around 2007, ref. 9) there has been a dearth of relevant proof of principle studies. I have not kept up with the field, but it seems to have shifted towards diabetes and mechanistic understanding. All in all, I do believe the field may deliver some breakthroughs, but it will take time. Meanwhile reduction of dietary AGEs may provide modest benefits (we don't really know).

I really fear that mediocre lifespan studies can stunt the development of whole fields, or as in the case of Resveratrol, completely derail the research. Multiple factors affect mouse lifespan and some are outside of our control (e.g. sporadic infections), but overall husbandry is often inadequate. Researchers need to stay strong and demand better mouse facilities!

1. Spindler SR. Review of the literature and suggestions for the design of rodent survival studies for the identification of compounds that increase health and life span. Age (Dordr). 2011 Mar 22. [Epub ahead of print] PubMed PMID: 21424790.

2. Nature. 2006 Nov 16;444(7117):337-42. Epub 2006 Nov 1.
Resveratrol improves health and survival of mice on a high-calorie diet.
Baur JA1, Pearson KJ, Price NL, Jamieson HA, Lerin C, Kalra A, Prabhu VV, Allard JS, Lopez-Lluch G, Lewis K, Pistell PJ, Poosala S, Becker KG, Boss O, Gwinn D, Wang M, Ramaswamy S, Fishbein KW, Spencer RG, Lakatta EG, Le Couteur D, Shaw RJ, Navas P, Puigserver P, Ingram DK, de Cabo R, Sinclair DA.
Problems: All mice unhealthy (fed a high-fat diet), no maxLS reported. It is not even so much a weak study; it's simply inadequate as an aging study.

Samstag, 20. Juni 2015

Optimizing resource use by outsourcing of lifespan research

Is it possible to outsource lifespan studies to laypeople (who are supported by professionals)? The idea may seem far-fetched to some of you, but in the end it would amount to a "pragmatic trial"; which is certainly an accepted study design. This type of study is generally less rigorous but considerably more realistic than a classic RCT (3).
It should be easy to get pet-owners to participate since life span testing on animals it at worst ethically neutral and most probably a net positive for the animals. Obviously, drugs and interventions designed to extend the lifespan will be minimally invasive and have a high risk/benefit ratio. Sine qua non. And since all animals age, enrolling them in such a trial would be beneficial for them; like enrolling cancer patients in a cancer trial.

Outsourcing of lifespan studies to pet owners
The idea seems ingenious and abstruse at the same time (1), but these days it has reached the mainstream. As we have discussed, Kaeberlein and others have suggested to test rapamycin for late-life rejuvenation in dogs. The advantage is obvious. As a researcher you can save on housing facilities, animal food, doctor visits and drugs (loving pet owners visit the veterinarian themselves), caretaker costs, taxes, toys, accessories, "environmental enrichment". On the other hand, you still have to pay drug costs, additional vet visits (e.g. specialized tests), recruitment, administrative costs and the salaries of the involved researchers. As it turns out, all the latter costs are quite high. Kaeberlein mentioned costs in the range of 10^5 to 10^6$for his study. Additionally, you have to cope with the added problem of heterogeneity (every pet owner behaves somewhat differently) and lack of training (these people are not trained technicians or health professionals).

Outsourcing of lifespan studies to zoos?
There are more than a thousand zoos world-wide by a conservative estimate (2), perhaps closer to 5000. I see no reason why those places could not conduct lifespan studies, if pet owners can. So far, I think I'm the first person to propose this study design. The idea is simple, one could for instance, mimic a cluster randomized trial (4). The advantages over individual pet-owners are lower heterogeneity in husbandry, availability of primates, better access to trained professionals, improved record-keeping, etc. It's difficult to think of a disadvantage, except that it's harder to recruit Zoos for altruistic reasons than pet-owners.

General ethics of animal keeping
There are concerns that keeping animals is unethical per se. (Some would even argue that all animal research is useless [5b]. They are wrong.) Husbandry is often criticized, especially in Zoos (5). I am sure this can be improved and it is necessary to do so. But is it unethical to keep well-husbanded animals? In the case of many animals the answer is, definitely not. The reason should be self-evident to any loving pet-owners and right now I don't want to delve into the details. Just to mention, an interesting argument for the ethics of keeping lab mice for longevity research is that they live longer, healthier and more peaceful lives in captivity than in the wild (6). At some point, I will probably do a longer post on research ethics in animal studies.

1. I became aware of this idea perhaps 5 years ago. It was championed (maybe even invented) by Edouard D. and other people from longecity/imminst.


3. Patsopoulos, N. A. (2011). A pragmatic view on pragmatic trials. Dialogues in clinical neuroscience, 13(2), 217.

4. Donner, A., & Klar, N. (2004). Pitfalls of and controversies in cluster randomization trials. American Journal of Public Health, 94(3), 416-422.

(not yet read in full)

6. Suckow, M.A., Stevens, K.A., Wilson, R.P., 2012. The Laboratory Rabbit, Guinea Pig, Hamster, and Other Rodents. Academic Press.
Note: The deer mouse (Peromysces), for instance, lives 8 years in captivity but only ~1 year in the wild.
I cannot find the data for M. musculus but it looks very similar from what I recall.

Samstag, 13. Juni 2015

What's hot in Aging Research? (2015 List)

I. Which interventions could produce a life-extension breakthrough in the next decade or so? (Short Term breakthroughs)
II. Which interventions hold promise, but are a little more speculative? (Mid Term breakthroughs)

Starting from now, I would like to review this question annually or every two years. In my analysis I'll include interventions, drugs, diets, etc. that could have a large and meaningful effect on aging or any of the major age-related diseases. From a methodological point, the answer must consider: A. effect sizes and B. plausibility (how advanced is the science?). Aspirin is an example of an intervention with high plausibility, but small effect size, since the effects are limited to cancer prevention. In fact, even if Aspirin reduced cancer incidence by 10-50% this would only result in 1 or 2 years added to the average human lifespan.

The answer to this question is personal opinion to some extent. To be as objective as possible I tried to track down reviews highlighting "important" research (3-6).

Mittwoch, 13. Mai 2015

The oxidative stress theory of aging - concepts and limitations

What do we know about the involvement of oxidative stress in disease?
Definitely not enough. The speakers at MiP-School London reminded us that researchers use the term "ROS" as explanation to hide behind with little explanatory value. Berry Halliwell (3,4) and Mike Murphy (6) gave fascinating talks on this topic. I would like to add a more biogerontologic perspective.

A deep mechanistic understanding of how ROS regulate lifespan might be called for, but I don't think we should over-emphasize detailed understanding in the field of aging as a whole. Gerontology is difficult. There are enough basic questions still unanswered. (Is calorie restriction a universal mechanism? Are current models of mouse lifespan extension redundant/convergent? etc.)

Historically, progress has come from rather crude phenotypic screens. Calorie-restriction was discovered before the Second World War, using mice without fancy molecular biology. To me, the whole Interventions Testing Programme is one big, naive phenotypic screen (readout = dead mice). The rationale for some of the tested compounds is almost cringe-worthy. Guess what? It's the most successful programme in the world, yielding multiple promising leads. Particularly, Rapamycin. To propose a test of rapamycin as a CR-mimetic we do not need to understand the underlying biology in any detail. Unsurprisingly, the first data on the mTOR pathway was generated using simple phenotypic screens in yeast and 6 years after the rapamycin lifespan study in mice, we still do not understand the mTOR pathway very well. The big invention by the ITP, and others like Spindler, were perfect animal husbandry combined with a modicum of understanding.

Given how little we understand about aging, the naive phenotypic screen will remain a worthwhile strategy. Knowing "just enough" is often quite useful. However, the naive approach to the oxidative stress theory of aging has produced conflicting results. Is it time to re-consider some of the basics?

Samstag, 4. April 2015

NIA's intervention testing program (ITP) - where does the journey take us now?

While this paper briefly outlines the history of the "intervention testing program", trying to find life extending drugs in mice, I want to comment on a tangential paragraph. As is known to anyone following the literature, the ITP identified rapamycin as the first drug to robustly extend maximum life span. Now the question arises how best to translate this data to humans? One important step will certainly be a long term study in long-lived animals.

And there exists an ingenious proposal for such a project (1, 2):

Matt Kaeberlein and Daniel Promislow at the University of Washington ... have been considering the potential value of a study in dogs living freely in the same environmental conditions as humans. They propose a small trial using 60 pet dogs that typically have life spans of 8–10 years, 30 of which would receive rapamycin and 30 would not. The trial would be started at 6 years of age, and the dogs would be monitored for cardiac function and other aging phenotypes such as development of type 2 diabetes and cancer until death. Such a study might reveal whether rapamycin retards aging per se, or acts principally by reducing late-life disease, but a larger study will probably be necessary to establish this unequivocally.

This basically outsources the lab work to pet owners. The idea is obviously brilliant, though, not completely novel. I have myself briefly collaborated on a project trying to get this done with mice. One of the biggest hurdles is getting enough people to participate - since such study must be large due to increased heterogeneity inherent to its design. Thus coordinating such a project through a university or some other body that is able to promote it makes sense. It goes without saying that large scale roll-out of this study type could save billions of dollars at virtually no cost.

The dog project by Kaeberlein is open to donations (3). Curiously, his wordpress page layout is even uglier than my blog. Unbelievable!

1. Age (Dordr). 2015 Apr;37(2):9761. doi: 10.1007/s11357-015-9761-5. Epub 2015 Mar 1.
NIA's intervention testing program at 10 years of age.
Warner HR

2. Hayden EC (2014) Pet dogs set to test anti-aging drug. Nature 514: 546


Samstag, 28. März 2015

Is complex I the culprit? Is it as easy as that?

Rapamycin, complex I and mitochondrial ROS
Despite plenty of controversy there is ample support for the mitochondrial free radical theory of aging. Here, I want to highlight some recent findings implicating complex I in mtROS production and aging.
Two recent papers (2, 4), Fok and Miwa, both find subtle reductions of specific complex I subunits after rapamycin treatment using *omic approaches. Miwa using proteomics and Fok using transcriptomics. Miwa et al. also showed decreased complex I and superoxide production after calorie restriction and reductions of complex I in a longer vs a shorter-lived strain. These changes were most pronounced in the matrix arm of complex I. Fok (4) studied chronic rapamycin feeding using transcriptomics ("Illumina Mouse Ref8 microarray") and found:
"The majority of the transcripts that change in complex I and II transcripts decrease while complex III, IV, and V transcripts that change increase in chronic Rapa-fed female and Rapa-2 male mice."

Mittwoch, 18. März 2015

Don't judge the primate studies of Calorie Restriction if you haven't read them!

I despair when non-expert scientists and intelligent lay people unknowingly spread misinformation about calorie restriction (CR). Not long ago I had to read again that the primate studies somehow refute CR. The reality, however, is more nuanced than that. Do not forget: misinformation can jeopardize public understanding and acceptance of research.

I will echo Ingram and Roth on this topic and use this post as a future reference. Both were involved (principal investigators, I believe) with the CR primate studies. (emphasis mine)
The long-term study of rhesus monkeys at the University of Wisconsin reported significantly reduced morbidity and mortality in those monkeys maintained on 30% CR from adult ages (7–14 years of age) compared to controls (Colman et al., 2009 and Colman et al., 2014). In contrast, a report from the long-term study of rhesus monkeys from the NIA that we initiated in 1987 found no evidence of improved survival in monkeys initiated on 30% CR from young ages (2–6 years) or older ages (14–21 years) (Mattison et al., 2012). Differences in the design of these studies, particularly the dietary composition, are now being investigated to uncover reasons for the different outcomes. Even if the conclusion is ultimately that CR does not significantly extend lifespan in rhesus monkeys, there is ample evidence from these studies to demonstrate improved health and function at older ages in monkeys on CR. 
Better yet not judge any of data, if your understanding comes only from popular science articles. I welcome comments by everyone, including lay people, as long as they are informed. I'll briefly mention another controversy for the sake of completeness:

Addendum: Does CR work in wild-derived or heterogenous mice?
This is important since these mice are more representative of healthy animals than, say, gluttonous black 6 mice. As in primates, I think the preponderance of evidence says "yes". A recent paper on the "yes"-side of the equation includes a review of other studies as well (2). This includes the negative study by Liao, Rikke et al. and Harper et al. with wild-derived mice.

1. Ageing Res Rev. 2015 Mar;20C:46-62. doi: 10.1016/j.arr.2014.11.005. Epub 2014 Dec 19.
Calorie restriction mimetics: Can you have your cake and eat it, too?
Ingram DK, Roth GS.

2. J Gerontol A Biol Sci Med Sci. 2010 Dec;65(12):1275-84. doi: 10.1093/gerona/glq155. Epub 2010 Sep 5.
Life extension by diet restriction and N-acetyl-L-cysteine in genetically heterogeneous mice.
Flurkey K1, Astle CM, Harrison DE.