Yamanaka Factors Turn Back The Clock For Mutant Mice
Could they do the same for humans?
By Flavie Prévost
In 2016, an experiment was conducted by researchers at the Salk Institute for Biological Studies. Its goal was to determine if the cyclic short term expression of the four Yamanaka factors in vivo would rejuvenate living breathing beings such as mice. Amazingly, it turns out it could. But how?
How did they activate the Yamanaka factors? Were there unwanted side-effects, effects we would want to avoid in humans? How long did the results last? Let’s explore the process behind the discovery that made countless sensational headlines a few years back.
(Warning: picture of mice internal organs in this article)
But First — What Are The Yamanaka Factors?
Yamanaka factors, which are named Oct3/4, Sox2, Klf4 and c-Myc (OSKM), are a set of genes that are highly expressed in embryonic stem cells, which completely dedifferentiate and get rid of any trace of aging. This is remarkable considering they come from gametes that have spent decades in another human’s body, which itself comes from the rejuvenated cells of their parents, and so on.
Over-expression of the Yamanaka factors by a cell transforms it back into its initial dedifferentiated, pluripotent and, most importantly, youthful state. However, de-differentiation and pluripotency are not the goal when trying to rejuvenate an organism, as we still want the cells to function appropriately. We want the eye cells to stay eye cells and liver cells to stay liver cells. This is sometimes tricky when experimenting with Yamanaka factors, as we’ll see here.
A Word on Progeroid Mice
The mice used for this study were not regular mice, as mentioned before. They carried a mutation that made them progeroid. But what does this actually mean? The definition of progeroid is “resembling premature aging”. That doesn’t get us very far. Why did these mice exhibit premature aging?
In fact, those mice, called LAKI mice, carried a G609G mutation in their “Lmna” gene. This mutation caused them to accumulate progerin, a truncated form of lamin A. This protein is responsible for the Hutchinson-Gilford Progeria Syndrome in humans, a syndrome that causes the people who are affected by it to show accelerated aging. Indeed, their average life expectancy caps at 13 years old. Furthermore, they are stricken by the same diseases that plague old age.
The choice is logical, then, to opt for progeroid mice to test aging reversal, as the results would be obvious. But this is where it gets interesting. The researchers wanted to use doxycycline to induce the expression of the Yamanaka factors. Doxycycline is an antibiotic of the group of tetracycline and, as we know, drugs can interfere with gene expression. For doxycycline to enable the expression of the Yamanaka factors, they decided to use a polycistronic cassette. A polycistronic cassette is a component of vector DNA comprised of a gene and a regulatory sequence we want the transfected cell to express. The trigger for the expression would then be doxycycline. The only problem was that those LAKI mice obviously did not come with polycistronic cassettes built into their cells.
To solve this, the researchers crossed LAKI mice with 4F mice. 4F mice do carry a polycistronic cassette containing OSKM, as well as a rtTA trans-activator. A rtTA trans-activator is what is going to turn up or down the expression of OKSM, or any gene, for that matter, specifically thanks to a drug belonging to the class of tetracycline (doxycycline in that case).
The researchers then obtained LAKI 4F mice, perfect to test their hypothesis.
Testing The Hypothesis
To verify whether cyclic short-term expression of Yamanaka factors resulted in improved age-associated phenotypes and extended lifespan, there had to be an experimental group and control groups.
For the control groups, the researchers didn’t only need LAKI 4F mice who wouldn’t undergo the rejuvenating treatment, aka the administration of doxycycline, as they already knew those exhibited accelerated apparition of a lot of age-associated physiological phenotypes, such as weight loss and modification associated with aging in multiple organs. Those mice were still needed, as they provided a sort of confirmation, but the researchers were going to need something else.
Thus, another control group was composed of LAKI 4F mice who would ingest doxycycline continuously instead of intermittently. Why is that? Well, the researchers wanted data for the cyclic expression of Yamanaka factors. Thus, they had to verify if continuous expression landed the same results. If so, the experiment couldn’t be conclusive.
The experimental group of LAKI 4F mice would ingest doxycycline through the means of their drinking water, 2 days a week, and drink normal water for the remaining 5 days.
What Happened To The Control Group?
As the researchers described it, continuous medication resulted in “significant weight loss” and, after 4 days, “high mortality”. Not exactly what we’re looking for. The researchers explained these results by the dedifferentiation that likely occurred in these mice’s vital organs, due to too high a dose, which turned back the clock on the mice’s cells back too far to their original state. Their organs then couldn’t perform their normal function anymore.
What Happened To The Experimental Group? — Physical Effects
(Warning: Picture of internal organs ahead)
The e3xperimental group started ingesting doxycycline at 8 weeks old. Throughout the duration of the experiment, which was 35 weeks, the following visible effects were observed.
- Maximum lifespan increase
- Median lifespan increase
- Reduction in spine curvature
- Improvement in the appearance of the gastrointestinal tract
- Increased epidermal thickness
- Increased dermal thickness
- Decreased keratinization of skin
- Decrease in tubular atrophy in kidneys
- Decrease in interstitial volume in kidneys
- Decrease of age-associated loss of parietal cells
- Reduced thinning of gastric epithelium in the stomach
In short, many symptoms of aging were successfully prevented. The only cloud on the horizon: LAKI 4F mice normally show a progressive weight loss associated with their accelerated aging. This symptom was not prevented. Still, this is an impressive improvement compared to control groups of LAKI mice, but also wild-type mice without a polycistronic cassette, on which the doxycycline did nothing.
What Happened To The Experimental Group — Cellular Effects
While the mice were busy not aging, a ton happened in them at the cellular level. Interestingly, the observed effects were all linked to the main pathways to aging and, spoiler alert: it improved them all. Let’s analyze each of the noticed cellular effects and see how they relate to the drivers of aging.
First, let’s take a look at DNA damage. The treatment significantly decreased the amount, intensity and volume of the spaces where a histone called γ-H2AX can develop. Why is that good? Histones are an important protein in the nuclei, and they make up a large part of the chromosomes. When spaces for the histone γ-H2AX develop, that means double-strand breaks have happened in the DNA, and it needs repair. DNA damage is one of the main drivers for aging, commonly comprised of genomic instability.
In addition, the amounts of the p53 binding protein 1 (p53PB1) in the mice’s cells were also decreased. One of p53PB1’s roles is to mitigate DNA damage, which isn’t surprising when we know that the consequence of a mutation in the p53 gene often results in cancer. Furthermore, the ingestion of doxycycline downregulated the expression of age-related stress response genes in the p53 tumor suppressor pathway. Examples of such genes are p16INK4a, p21CIP1, Atf3, and Gadd45B. This means the organism had to work less to suppress imminent tumors because there were fewer of those. Fewer cancers are a phenotype associated with youth.
Finally, improvements were noticed in the cells’ heterochromatin. The heterochromatin is responsible for supporting and protecting the chromosomes, thus preventing genetic material damage. The treatment upregulated the activity of H3K9me3, which is normally downregulated with aging. Plus, it downregulated the activity of H4K20me3, which is, you guessed it, upregulated during aging. Both H3K9me3 and H4K20me3’s normal activity are essential for proper heterochromatin maintenance.
The treatment also improved the mice’s health relating to senescent cells. Indeed, the senescence-associated metalloprotease MMP13 and interleukin-6 were downregulated, which means fewer senescent cells were created. Senescent cells are generated as a means of the organism to protect itself. When a cell’s DNA is damaged, it can die, become cancerous, or become senescent. Ideally, the body uses autophagy to kill those damaged and potentially dangerous cells, but as it ages, it progressively loses its ability to do so. Thus, it makes them senescent, which is less dangerous than letting them become cancerous, at least in the short-term. Another senescence-related observed effect was the reduction of senescence-associated β-galactosidase activity.
Third, let’s talk about reactive oxygen species (ROS). ROS are produced when damage has occurred in the body, as I explain further in this article about the mitochondria. The doxycycline treatment reduced the production of ROS in the cell by the mitochondria. This means probable lessened mitochondrial damage, as well as lessened overall stress in the cell.
Lastly, LAKI 4F mice, due to their progeria, typically accumulate progerin in their nuclear envelope, thus degrading it. This is a driver of premature aging in these mice, in humans with Hutchinson-Gilford Progeria Syndrome, as well as in regular aging. Thankfully, the induction of OSKM in LAKI 4F mice’s cells significantly improved their nuclear envelope architecture compared to untreated LAKI 4F cells.
But Doesn’t Yamanaka Factors Expression Lead To Tumors?
Tumor development when manipulating the expression of the Yamanaka factors is always a concern because one of them, c-myc, is an oncogene. However, the gradual and measured expression of OSKM resulted in the disappearance of the aging phenotype in LAKi 4F mice without loss of cellular identity. Since these mice also didn’t develop tumors, the researchers hypothesized that this gradual method could potentially also be responsible for the prevention of cancer as an unwanted side-effect of this treatment.
How Long Did The Results Last?
Good news or bad news first? We’re going to go with the bad news: 4 and 8 days after treatment termination, the LAKI 4F mice’s cells were analyzed to see if at least part of the aging phenotype had been recovered. And sadly, this was the case.
Now, this might seem like a short time, but we have to remember that mice, especially LAKI 4F mice, have a much shorter lifespan than humans. In addition, the duration of the effects of the doxycycline treatment is likely to be proportional, just out of sheer logic. Thus, 4 to 8 days in mice might represent much more time in humans.
There is another good news. 4 days after these observations, the researchers re-administrated doxycycline, to see if this newly acquired aging phenotype was also reversible. It turns out it was.
A Final Word…
Let’s review a few key results that should ring a bell. First, the researchers observed significant improvement in multiple pathways to aging, meaning many drivers of aging were reduced, thanks to one single intervention on the Yamanaka factors. Second, even if the treatment wasn’t permanent, it was re-administrated with success. What gives?
Could we possibly have found a single silver bullet to preventing aging as a whole? As always, probably not. More studies will be necessary to tell if this is the case. For example, we’ll need an efficient way to express the Yamanaka factors in humans. We don’t come with OSKM polycistronic cassettes, after all.
But the results of this study sure feed hope for fighting aging. Maybe one day, not too far into the future, we’ll all regularly go to a clinic to get our internal clock reset, or even just take a pill for the same effect, and enjoy life fully as we once could, as long as we live.
