Old mice have rejuvenated their eyes

Reprogramming restored visual acuity to elderly and injured mice

Polina Loseva, N+1

Retinal neurons damaged by glaucoma were forced to regenerate for the first time. This result was achieved by American biologists using partial reprogramming technology: they injected viral vectors with Yamanaki factor genes into the eyes of mice, which did not transfer neurons to the embryonic state, but restored their operability. The same thing was done with aging animals – Yamanaki factors returned them to their former visual acuity. This study is published in the journal Nature (Lu et al., Reprogramming to recover youthful epigenetic information and restore vision).

Apparently, demethylation is responsible for the rejuvenating effect here: under the influence of Yamanaki factors, a "young" set of epigenetic tags on DNA is restored in cells. But how the cell "remembers" which set of labels corresponds to its youth is still completely unclear.

One way to distinguish old cells from young ones is to look at a set of epigenetic markers, that is, methyl labels on DNA and histone proteins on which this DNA is wound. Over time, some genes acquire these labels, while others lose them. Thus, the set of genes that are available for reading information changes in the cell, and because of this, an aging cell loses various properties inherent in a young one - for example, the ability to divide, turn into other cell types or get rid of molecular debris. Therefore, a set of tags on DNA is the so–called "epigenetic clock" – can be used to measure a person's biological age.

There are still no working methods to reduce it (except for one case when participants in a clinical trial aimed at restoring thymus function became two years younger epigenetically during the year of the study), but it is possible to "reset" the epigenetic age for individual cells. This can be achieved with the help of reprogramming technology, for which Shinya Yamanaka received the Nobel Prize in 2012. Its essence is that four transcription factors (Yamanaki factors) are introduced into the cells, which transform them from differentiated into stem cells, similar to cells in the early stages of embryogenesis – and the epigenetic age also begins to correspond to the embryonic state.

It is much more difficult to apply this technology to the whole organism. Firstly, in order to rejuvenate the body, it is not necessary to turn its cells into germ cells. It would even be harmful – tumors can grow out of them, and they will cease to perform their functions at the same time. Partial reprogramming could solve this problem – for example, when transcription factors are injected into cells for a short time.

The second difficulty is to act on all the cells of the body at the same time. This result was achieved in prematurely aging mice, whose life was extended by 30 percent with the help of reprogramming. But to do this, the mice had to be genetically modified – that is, to embed the genes of Yamanaki factors into the genome of mice so that they could be triggered by a signal from outside.

This technology is unlikely to work with humans – at least, it is forbidden to genetically modify them yet. Therefore, a group of scientists from Harvard Medical School, which included well-known biologists Vadim Gladyshev and George Church, gerontologist David Sinclair and developers of epigenetic clocks Morgan Levine and Steve Horvath, decided to try a different technique.

The researchers created an adenovirus vector that was supposed to deliver the genes of three Yamanaki factors into mouse cells (the fourth, as the most oncogenic, was decided to exclude). This makes it possible not to modify the whole organism, but to introduce genes into individual cells under study. At the same time, all three genes were under the control of the promoter, which is triggered only by the action of the drug doxycycline. Thus, the authors of the work were able to start and slow down the work of Yamanaka factors from the outside – simply by adding doxycycline to drinking water for animals.

To begin with, the researchers checked that this technique was safe: they injected their vector into both young (5 months) and elderly (20 months) mice and activated the Yamanaki factors from time to time, but did not notice any differences from the control animals in health status or in the number of tumors.

Then the authors decided to test their method of cell rejuvenation on retinal ganglion cells. These are neurons, through the processes of which nerve impulses from the eye enter the brain. They are notable for being able to regenerate and grow new axons in embryos and newborns, but lose this property with age – and therefore damage to the optic nerve, for example, in glaucoma is irreversible.

Drawings from the article by Lu et al. The mice were first injected with viral vectors inside the eye and triggered the work of Yamanaki factors with doxycycline, then the optic nerve was damaged, and then dyes were injected that marked the growing axons.

It turned out that under the influence of Yamanaki factors, the number of regenerating axons increases almost 5 times – but only when the vector contained all three factors. Singly, they had a much weaker effect. Moreover, it was possible to start regeneration both in the young (1 and 3 months) and in the elderly (12 months) mice. Similar results were obtained by the authors of the work on human neurons – however, only in vitro.

The number of regenerating axons in the optic nerve of mice after injury. The blue line corresponds to the expression of all three Yamanaki factors.

After that, the researchers tested whether it was possible to restore vision in the damaged eye in mice in this way. Microgranules were injected into the anterior chamber of the eye to increase pressure and cause glaucoma. After that, adenoviral vectors with Yamanaki factors were injected into the vitreous body and their work was started with doxycycline for 4 weeks, and then the visual acuity of mice was measured. During a month of reprogramming treatment, the researchers managed to "win back" about half of the loss of visual acuity in mice – and, it seems, this is the first example of vision restoration after nerve injury in glaucoma.

Visual acuity of mice before (left) and after treatment (right). Gray column – mice were injected with saline (control), orange – microgranules (which cause glaucoma), red – microgranules and viral vector (without activation), blue – microgranules and viral vector, activation by doxycycline.

Finally, they tested the same technique on aging (11 months) mice whose visual acuity decreases even in the absence of glaucoma. It turned out that the introduction of Yamanaki factors in them also improves vision – and so much so that it becomes indistinguishable (p = 0.814) from the vision of young (3 months) mice. And this already means that reprogramming can be used to reverse age-related changes, even if they are not related to a specific disease. And theoretically, this technique could work in humans as well – since the eye is separated from the main blood flow by strong barriers, and adenoviral vectors injected into the eye may not get into other organs (therefore, it was in the human eye that the implantation of reprogrammed cells, as well as genetic editing in vivo, was first tried).

Visual acuity of young (left) and elderly (right) mice before (red) and after (blue) administration of Yamanaki factors.

Then the authors decided to find out how DNA methylation changes during these processes. After measuring the epigenetic age of neurons by a set of tags on ribosomal genes, they noticed that the injury of the optic nerve increases the age of cells, and the work of Yamanaki factors, on the contrary, reduces it. At the same time, when the work of demethylases – proteins that remove tags from DNA – was blocked in neurons, they could not regenerate. This means that in order to restore the ability of nerve cells to grow axons, it is necessary to reduce their epigenetic age.

However, in the experiment with aging healthy mice, there was no eye injury, and the nerve cells did not need new appendages. It means that there Yamanaki factors and demethylation were able to change something in the very physiology of cells and return them to their former working capacity. Indeed, the researchers calculated that the expression of 464 genes changes in aging ganglion cells, and 90 percent of them return to the "young" level after the introduction of Yamanaki factors.

Thus, it turned out that reprogramming cells can somehow return them to a young state without turning them into stem cells. At the same time, they do not lose their previous functions and performance, but "reset" their epigenetic labels on DNA. But as soon as the cell can return to the "young" epigenetic state, this means that it must be "recorded" somewhere. The authors of the work note that they do not have an answer to this question, but there are only assumptions – that this "initial state" can somehow be encoded by other DNA modifications, chromatin changes or DNA-binding proteins. But if it can be detected and decoded in one way or another, then perhaps we will learn how to reduce epigenetic age in other tissues and organs, and not just in individual neurons of the eye.

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