The mouse that time forgot

A year ago, it hit the world’s headlines: Dutch researchers had developed a substance that makes mice not older – but younger. Have these scientists finally cracked the code of old age, and found a potion that can reverse aging? For a year, reporter Maarten Keulemans went behind the scenes of the crucial experiment testing the 'miracle drug' called FOXO4-DRI.

Maarten Keulemans
null Beeld Rein Janssen
Beeld Rein Janssen

April 7, 2017

115 weeks

Age in human years: 80

An iron fence. Keycard. A door. Keycard. Put on lab coat, mouth cap, blue latex gloves. Hallway, yet another door.

And there they are. The mice that defy time.

Mouse 15-MI10959-05F wakes up in a bad mood. Just moments ago, it was still fast asleep in a tuff of cotton that all lab mice have in the transparent plastic containers they live in. And now this: these huge, blue hands. The tiny little claws grope around and clasp to the lower arm of PhD-student Marjolein Baar. At lightning speed, the little nose ferrets up and down. ‘Look how agile and fit it still is’, Baar says, looking at the little female mouse climbing up her lab coat. ‘She is all over my sleeve. Even though these mice are ridiculously old. One hundred fifteen weeks.’

That’s something like 80 years, loosely converted to human lifetime. Not exactly an age at which a human being still briskly goes out for a climb, to explore what’s going on.

All in all, there are six mice like 05F. Three of them get a monthly jab with saline solution – the control group. And then there are three mice that are treated once a month with Proxofim. That’s a compound that made world headlines in March last year because it, so the story went, had made mice biologically younger.

And now, the follow-up experiment is ongoing. Six reasonably ordinary mice – ‘reasonably’, because the mice have been genetically altered a bit so that certain cells in their tissue become visible – that are injected either with the placebo or with Proxofim, to test whether the remedy is safe. And to see how old the mice become, even though that’s not the study’s main purpose.

null Beeld Arie Kievit
Beeld Arie Kievit

Baar notes a small, cardboard label that dangles down from one of the containers. ‘BIG’, an analist has jotted down there. Inside the cage, there’s indeed a rather corpulent mouse. ‘Big? Look at you’, the label reads on, in another hand. ‘Signed: the mouse.’

Five floors up, in the research group’s work space at Erasmus Medical Center, the atmosphere is leisurely. The past few days, the Rotterdam group found itself in the eye of a true media storm, following the release of its new paper in Cell. Research leader Peter de Keizer, a talkative scientist in his thirties with a cheerful twinkle in his eye and a thin beard, appeared on several Dutch news shows, and was interviewed by news media ranging from Science in America to Cosmos in Australia, and from the BBC in the United Kingdom to The Daily Star in Arizona. De Keizer, the man who ‘can restore hair and give youthful energy’, as the British tabloid The Daily Mail phrased it.

Well, De Keizer. ‘BAAR et al.’, first author Marjolein Baar has jotted down on the group’s whiteboard, in teasingly oversized capitals. That, at least, is the formal quotation of the scholarly article the fuzz is all about.

The room is vibrant with creativity, and full of young people. ‘That’s what you think’, De Keizer says. With a broad grin he addresses Hester van Willigenburg, a young PhD student who has just entered the room. ‘You’re in your seventies, aren’t you?’

May 12, 2017

120 weeks

Age in human years: 84

In his teenage years, De Keizer had wanted to become a veterinarian. But that was before he fell under the spell of the cell. That tantalizing, tiny building block that all earthly life is made of, packed with proteins and other amazingly complex molecules. Breathtaking. Fascinating.

So a degree in biology, at Utrecht University, it became. He obtained his doctorate with a thesis on a promising molecule that drives certain cancer cells to suicide. Got married, and moved to Novato, California, where he was a researcher at the Buck Insitute for Research on Aging for two and a half years.

And now he is in Rotterdam, gently poking a finger into a mouse’s belly, saying: ‘You little fatso. It’s just fat you have here. Fortunately, no tumors.’

Peter de Keizer enjoys the media attention. Fantastic that a molecule you single-handedly devised, actually seems to be doing something inside the cell, as he puts it during one of our meet-ups. Proxofim – ‘FOXO4-DRI’, in molecular lingo – is already the third version of his anti-aging molecule, after two earlier ones he developed in the US. And now, FOXO4-DRI is abuzz with research activity: Baar helps with the molecular research, Van Willigenburg explores if the molecule has any use after an organ transplant, another PhD student named Diana Putavet investigates if the compound has effect on boldness or certain cancers.

For the mice in the basement, the years are beginning to take their toll. Especially 15-MI10959-05F, the untreated female mouse that was still lively climbing around Baars arm last time, is going downhill, De Keizer says. He takes the little rodent by the scruff of the neck and gently feels its belly, while the animal willingly leans over. ‘I think she has tumors’, he says. ‘I’m afraid it won’t be long before we have to call the vet.’ If the discomfort becomes too much for the animals, they are taken out of the test, as it is called.

Old age. Wouldn’t it be great if we could do something about it?, De Keizer muses. With the counting of the years come the ailments of old age, and it are those infirmities that can make life a misery and that put society to expenses. Rheumatics. Cardiovascular diseases. Cancer. Dementia. ‘People retire. And a dozen or so years later, decline begins’, De Keizer says. ‘If only you could slow down that process. So that you become sick later, have more years in good health. That’s what we’d like to accomplish most of all.’

null Beeld Rein Janssen
Beeld Rein Janssen

That’s less absurd than it sounds. Over the last decade or so, scientists have discovered a myriad of compounds and substances that keep aging tissues young, for example because they put cells in maintenance mode or mimic the beneficial effects of exercise or rigorous fasting. That already led to a conspicuous parade of extremely long living lab animals and, coming soon, the cautious, first clinical trial on aging American men and women, using the drug metformin.

De Keizer targets the so called ‘senescent cells’. Those are body cells that have stopped dividing, but that still secrete all kinds of infectious signals and other spoiling substances. The older we get, the more senescent cells pile up in our tissues. It’s one of the main drivers of the signs of old age, a team led by Jan van Deursen of the Mayo Clinic in Rochester, Minnesota revealed in a groundbreaking series of studies around 2010.

So better begone, senescent cells. About twenty compounds are known to break the cells down – but most of them have eerie side effects, or are useless for other practical reasons. And then there’s FOXO4-DRI: a simple protein fragment called a ‘peptide’ that gently prompts senescent cells to commit suicide.

The first series of mouse tests – the one by BAAR et al. – proved quite promising. The treated rodents had more fur, better kidney function, and seemed fitter. But the mice were sacrificed for science prematurely, in order to study their innards. The mice he’s investigating now, are allowed to age normally.

‘Three per group is of course not enough to draw any firm conclusions’, he emphasizes. ‘But on the other hand: if all treated mice are better off, it’s surely hopeful.’

June 14, 2017

125 weeks

Age in human years: 88

‘Someone’s using this on himself already, did you know that? To slow down aging. And only this morning I’ve received yet another e-mail of someone saying: I consider using it. Well, what can I do about it? Call the cops?’

Peter de Keizer scowls over his green mouth mask. A few minutes ago, he took out his smart phone to show me a Chinese website where the Rotterdam compound is already for sale, for several thousands of dollars per cure. That’s the way it goes, he explains. ‘Everyone can copy it, the details of our peptide are in our paper. There’s a demand, so labs supply it.’

Better watch out, De Keizer keeps telling them. ‘You just don’t know what kind of risk you take if you inject this. Suppose you’re 70, and you have all these senescent cells in your brain: will it destroy your memories then? Have you thought that over?’

The pleas from desperate, terminally ill patients – those are the ones that move him most. Ever since De Keizer noted, on television, that his drug may also affect certain types of cancer, patients know how to find him. ‘The other day, I got an e-mail by someone terminally ill with end stage glioblastoma’, he says. Suddenly, there are tears in his eyes. ‘The guy was 31, had a family, two kids. Just like myself. I can tell you, that really hits home.’

Still, he has to say ‘no’, in cases like that. ‘From a human perspective I would say: sure, give it a go. But I’m not a medical doctor, I don’t even know if this is the right molecule. And besides, I’m punishable by law, should I provide it.’

null Beeld Arie Kievit
Beeld Arie Kievit

There’s a friction here, he says. ‘The traditional route, of years and years of testing on mice, simply takes too long. I think we should start clinical trials much sooner. Especially when it comes to patients who only have a short time left to live, and who request for this themselves.’

Not to mention the elderly who buy the stuff over the internet, for themselves or even – yes, they send him e-mails about that too – for their pet. ‘People just don’t take it anymore having to wait so long. And you can’t blame them. If you’re 75 years old, you simply don’t have the time anymore. So they purchase it themselves, because they’re old and they have the money. The current medical system is absolutely unprepared for that.’

July 6, 2017

128 weeks

Age in human years: 90

‘I think it was a heart attack’, Diana Putavet says. ‘Something sudden, in any case. It was just sitting there, in the corner of its cage. And then it suddenly stopped moving.’

A little over 2,5 years old are the mice now, and after two of the mice treated with salt solution passed away – the obese one and the sleeve climber – the first treated mouse has died, too. ‘They are getting older’, Putavet, a small, Rumanian born PhD student, observes. ‘Their diseases begin manifesting.’

Even immortality apparently has its limits. This is how it usually goes in medical research: on closer inspection, the truth often turns out much more balanced than the miracle stories from the media had led you to believe.

Just wash away the cells of old age, and presto – you’re young again? Forget it. In a body that ages, there’s more going on. The DNA wears out, and becomes prone to cancer. The rejuvenating ‘stem cells’ run out. The energy supply of the cells starts hampering. ‘There are seven pillars of aging, and we’re targeting just one of those pillars’, De Keizer says. ‘In the end, you’ll always have to do multiple things. Eat less, exercise more. Train your muscles, maintain your supplies.’

But what about those mice we saw on TV: one rodent visibly old and covered in bold spots, the other treated by De Keizer and reasonably all right? These were special research mice, De Keizer explains, genetically engineered to age super fast. ‘I’ve always said this. But you know, on TV, distinctions like that tend to drop out.’

Well, at least there are still two Proxofim treated mice left – and they’re doing fine. Carefully, Putavet lifts mouse number 15-MI10957-07F out of its container and puts it on the table. Heave-ho, immediately the animal goes out for a stroll.

‘It isn’t forty anymore. But it’s still walking cloppety-clop’, De Keizer observes.

‘It looks like a mouse half a year younger’, Putavet says.

October 5, 2017

141 weeks

Age in human years: 99

On our way to the mouse lab, Peter de Keizer suddenly starts singing. Elton John. ‘I’m still standing, yeah yeah yeah.’

For it’s true. The last few weeks, two more mice have died, one treated and one from the control group, but mouse 15-MI10824-03M is still standing. Although you can leave out the ‘yeah yeah yeah’: the little mouse is trembling some, one of its eyes is a little infected and it walks a bit straddle-pawed, with old mice’s steps. Time is ticking, even for a mouse that tries to live forever.

‘Still, it looks much better than many of our younger mice’, Putavet thinks.

‘It’s a bit grey. And it walks a bit like that, you see?’, says De Keizer.

‘That’s what you see in older people. They walk a bit funny’, Putavet says.

‘I don’t think it will die over the next couple of weeks’, De Keizer says.

If only 03M knew. Over the past few weeks, a lot has happened upstairs. The university joined forces with a new company, especially founded for De Keizer’s peptide, Numeric Biotech – the word ‘numeric’ refers to the numeric age Proxofim is supposed to stretch. And now Numeric wants to prepare the molecule for the first rounds of clinical testing, in patients with certain types of cancer.

But Peter de Keizer himself opts out. De Keizer bites his lips, he’s not allowed to discuss it, ‘that is what we agreed on’, he says. But insiders, who only wish to speak to me on the condition of anonymity, sketch what has happened: a hell of a row. De Keizer had wanted to coach the peptide to the first patient trials himself but thinks the molecule isn’t ready yet and didn’t want to hand it over; Numeric on the other hand finds De Keizer too buttoned-up and has pressed him to cooperate.

It’s a row the parties involved decline to discuss in public. ‘We’re glad with the partnership with Numeric Biotech, because this pushes forward the research’, Erasmus MC reacts in a statement. ‘What Peter has decided, is up to him’, project leader Brian Eisenburger of Numeric says over the telephone. ‘As far as we’re concerned, we’re open for all collaborations.’

null Beeld Rein Janssen
Beeld Rein Janssen

But De Keizer leaves. To Utrecht University, his old home ground, and Putavet and Baar are joining him. Out to look for a revised version, a new peptide to counter the burdens of aging. The FOXO4-DRI patents stay behind in Rotterdam; Erasmus MC has the property rights. For De Keizer, it’s a tremendous blow, insiders explain. To know that ‘his’ peptide, which his group had already jokingly given the pet name Proxofim, is not really his in the end – but his employer’s.

If only mouse 15-MI10824-03M knew, in its basement. A bit unsteady, it struts about the table, a tiny old mouse of about 99 years old, if you were to put it in human years. ‘Of course, this means nothing in itself. It’s just one mouse’, De Keizer stresses. ‘But having said that – it also doesn’t mean the opposite is true.’

January 23, 2018

157 weeks

Age in human years: 110

An iron fence. Keycard. Put on lab coat, mouth cap, blue latex gloves. Hallway, yet another door.

And there it is. The mouse that time forgot.

‘You see? It really is still alive’, De Keizer says.

‘Look at that. It’s looking for food. That’s a very good sign’, Putavet remarks.

Mouse 15-MI10824-03M turns out to be a tough one. The back somewhat crooked, the fur a bit rough, its ears kind of rumpled. But once released out of its housing, the old chap still strolls around just fine, the little tiny little nose curiously sniffing like old times. ‘YES’, the label at its container reads. That indicates that this is a treated mouse – but one could easily read it as a shout of joy.

‘A mouse that lives this long. I don’t buy that it’s just coincidence’, De Keizer says. Putavet sums up the statistics: in the Rotterdam facility, usually only one out of three mice survives beyond two years, and 140 weeks, that’s a lifespan given to only very few mice. ‘And now we’re at 157 weeks. That’s an incredibly long period. I think it’s a first, for this facility.’

Once a week, Putavet drops by to inspect the miracle mouse. The Rotterdam group is disbanded by now. De Keizer and Putavet work in Utrecht nowadays; Baar and Van Willigenburg will presumably follow later. A follow-up experiment, with another group of six mice, was cancelled; 03M truly is the last one of its kind.

And now the mouse that was allowed a taste of the fountain of youth is pottering about the table, while De Keizer affectionately addresses it: ‘You little old rascal. We’ve solved aging, how about that?’

March 9, 2018

163 weeks


Happy birthday to you happy birthday happy birthday – and so it turns out that Marjolein Baar, Diana Putavet and Peter de Keizer are shaking hands in the coffee corner of their new building at the University of Utrecht. Still a bit ill at ease, because to be honest, they don’t really know the birthday boy – an Asian PhD student named Can – that well yet. ‘There’s always something to celebrate here’, De Keizer tells me. ‘Almost every week someone is organizing a drink, to toast to the acceptance of their new paper.’

By now, the team is almost completely relocated to Utrecht and is making a new start. ‘Numeric chooses to continue with this project, and we don’t’, De Keizer says in retrospect. ‘I think the peptide isn’t safe enough yet.’ The molecule is still ‘too heavy-handed’, he goes on to explain: for every ten senescent cells, it also drags one healthy cell into death, and that’s a ratio that he’d like to improve to a hundred to one.

‘The concept still stands’, De Keizer says. ‘Peptide number three was good enough for mice. But now it’s time to move on to peptide number four, five and six. Perhaps peptide number six will be the one we’re looking for.’

And yes: together with his coworkers Marco Demaria of the University of Groningen, Tobias Madl of Medical University Graz and biotech enterpreneur James Peyer, he has founded a company of his own, Cleara Biotech, to hedge the next version of Proxofim commercially. No more of what happened to him in Rotterdam. ‘We’d like to leave that behind us now’, he says. ‘A closed chapter. We’d rather look to the future.’

That’s a future Numeric is also looking to. ‘It is our intention to bring this molecule to the clinic somewhere over the next one or two years, for certain indications in the oncological field’, Eisenburger says. It needn’t be a problem that the substance still isn’t very precise, he thinks: ‘That’s an assessment one should make depending on the situation: for which medical indication do you intend to apply this, what dosage do you use?’

null Beeld Rein Janssen
Beeld Rein Janssen

A long and healthy life is not among the medical conditions Numeric want the peptide tested on. If only, because old age officially just isn’t a medical condition, Eisenburger explains. ‘We’d love to do it. But as it is, you just won’t get it funded.’

Down in the basement of Erasmus MC, in its secured lab, 03M has now passed away. Quietly and without much ado it went: the mouse had become too old and had, according to the guidelines for animal welfare, reached its ‘humane endpoint’. So it was lifted out of its cage, gently put inside a specialized machine and quietly soothed to sleep with CO2. An animal friendly end, neatly according to protocol – De Keizer and Putavet weren’t even present.

And now, it is in the freezer, the last tangible memory of the adventure with Proxofim for the moment. A tiny, black mouse, solidly frozen, the back crooked with old age, its fur flocky and dull.

Three years, one month and one week old, 03M eventually became – that’s 1133 days, and sort of a record. According to the official statistics, the type of mouse of which 03M is one, usually doesn’t ever get older than 1000 days. But 03M became 113 years, loosely calculated in human time. By all means, a quite respectable enviable age.

‘Whether it takes two years or ten’, De Keizer says, ‘eventually we will have something we can apply on humans. Of this I am fully convinced.’

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