This article is based on a poster I presented on “Forschungsforum HTW Berlin” in October 2021. The poster is available in German language on DOI: 10.13140/RG.2.2.26595.43048.
What’s so cool about Telomeres?
Telomeres are the Ends of Chromosomes and shorten with each cell division. Once they reach a critical short point the cell becomes senescent. Senescent cells are responsible for the aging of organs. On the other side Telomerase can stabilize or elongate Telomers. It is active in the germ line, stem cells and in about 90 % of all cancer cells. This makes Telomer dynamics an important target for aging and cancer research.
Telomeres are relevant to both aging and cancer. Drugs elongating or stabilizing them could delay aging. Drugs that shorten them could be used in cancer therapy.
Project Idea
Drug development that affects Telomere length and dynamics requires systems to screen for these parameters. In such a system the influence of thousands of substances on Telomeres has to be examined in parallel. It has to be cheap, quick and easy.
Idea: A high-throughput system that uses optical signals to estimate the telomere lengths of living yeast cells. For this purpose, proteins that naturally bind to Telomeres will be labeled fluorescently and/or luminescently.
Problem: Yeast use Telomerase to keep their Telomeres constant. In addition to the marking the Telomers, I need to integrate a switch on the Telomerase of the yeast to activate and deactivate it.
Markers for Telomer Length
Schematic representation of a Telomere and the main proteins associated with it. The three proteins I’m using as markers are marked with green lightbulbs: Rap1 and Rif1/2. Their close association on the Telomer should make it possible to use molecular distance measurement methods like Quenching, FRET and BRET (see below).
Methods for Marking
The marking is done in the genomic code of the target proteins (RAP1, RIF1 and RIF2) by fusion with reporter proteins. The genetic code of the reporter (a fluorescent or luminescent protein) is attached to the code for the target protein in the genome by means of transformation and recombination.
Possibilities for labeling the proteins: Fluorescent markers light up in a different (red-shifted) color when they are illuminated, bioluminescent markers light up when a substrate is converted, two identical fluorescent markers reduce their fluorescence when they come close to each other (quenching), a fluorescent marker can transfer energy to another, which then glows in a different (even further red-shifted) color (FRET), a luminescent label can transfer its energy to a fluorescent label, which then glows in a different color (BRET), these methods allow distance measurement at atomic scale level
Methods to turn Telomerase on/off
In order to make the Telomerase switchable the promoter of one of the genes that together form the Telomerase is exchanged. A promoter is a regulatory sequence in front of the actual gene (the blueprint of a protein). It controls how and when the encoded protein is produced.
The natural promoter of the EST2 gene, which is one of the components of yeast Telomerase, is replaced with the promoter of the DDI2 gene. While Telomerase is always active in yeast, the DDI2 gene is only activated under certain chemical stimuli. If the promoter is successfully exchanged the Telomerase is inactive but can be activated by adding small amounts of the chemical cyanamide.
One way to achieve this is to exchange the promoter directly in the yeast genome via recombination.
Another slightly simpler method would be to destroy (knock-out) the gene in the yeast genome and introduce a functional version with the exchanged promoter on an artificial chromosome (plasmid) into the cell.
Status of work after year 1 of 4
Libraries of yeast strains with GFP-tagged proteins are available. These were used to carry out the first preliminary tests to establish the measurement methods.
A DNA construct was designed and produced that makes it possible to replace the promoter of the EST2 gene. The construct was transformed into strains with different GFP tags.
Transformed colonies are currently being examined to determine whether and where the construct is integrated in the genome. It is also checked at the protein level whether the control works. This has just been confirmed by a Western blot which I will explain in one of my next articles.
Next the new strain will be tested in the fluorescence assay.
The good news is, slowing aging and gaining more than ten years is absolutely possible and doesn’t cost you anything except some discipline (yeah, I know, that’s the bad news here). However, to keep you reading, I’ll tell you a little later how that works. Whether it is possible to slow or even reverse aging is currently the topic of a lot of research. But let’s start at the beginning. If you want to treat something, you first need to define it clearly. The most obvious definition for aging is chronological. However, changing the actual flow of time falls more into the realm of physics and is probably not very practical. What we want to influence is the biological age. To measure this, we use a lot of different methods, called “clocks,” and they work with blood parameters, heart rate variability, epigenetics, simple photos of your face, or other data. Clocks are all somewhat linked to chronological age but can generally tell you how one (or several) aspects of your biological age compare to the average person your age. So they tell you if you’re younger or older than you actually are.
The specific unpleasant cellular effects of aging are summed up as the 9 Hallmarks of Aging that you see above. I won’t go into detail, but they are all interconnected and lead to what we recognize as aging, like wrinkles, grey hair, loss of muscle mass, frailty, dementia, decreasing bone density, and all the other stuff that you’re not keen on having.
Reading this list, you might already guess why treating aging might have other perks than just living longer. The biggest deal, not only for the individual but also for society, would be increasing the so-called healthspan. It can be argued that while in the last 100 years we have already more than doubled the average life span, the healthspan, the time lived in good health, hasn’t grown accordingly. Our current medicine has become very good at treating most of the countless ailments that old age brings; however, many are more managed than cured. So wouldn’t it be better (and cheaper, by the way) to treat the underlying cause of most illnesses instead of each of them at a time? The results of healthspan research could revolutionize medicine and bring us from fixing what’s broken to preventing the breaking.
Lifestyle Interventions to Slow Aging
But how far along are we? Will we still get old like our grandparents? That depends. To cite one of the leading minds in this field of science, Professor David Sinclair: “It’s easy to expand your lifespan. […] If you do the right things, which is: Don’t overeat, eat less often during the day, do some exercise, don’t smoke, don’t drink! That alone gives you, compared to people who don’t do that, 14 extra years. So living longer isn’t hard, it just takes some discipline.” Well, I told you, it’s not too easy, but it’s doable.
Especially the eating less often part seems to be important. Intermittent fasting (best more than 16 hours without food) gives the cells a feeling of food scarcity and switches on certain survival programs. Probably the most important is autophagy which let’s cells recycle accumulated proteins and other reserves. This kind of a cleaning helps get rid of things that can cause trouble when they accumulate too much.
The other main effect is a reduction in metabolism and especially on cell division. Since cell division is on multiple levels the main reason for mutation (errors in the DNA) it is also the main reason for aging. Avoiding strong mutagens like smoking, excessive drinking (one drink a day seems to be positive) and sun bathing is helpful for the same reason.
Enough sleep and some exercise have also been shown to positively affect aging in human studies.
However, there is obviously more to aging research than the typical advice on living a more healthy lifestyle.
Different times for intervention to delay illness and death by delaying aging, Figure CC from Douglas R Seals, 2014
Supplements and Drugs
First of all, there are drugs and supplements that (at least in animal models) show a huge potential to give another few healthy years like Nicotinamide Mononucleotide (NMN), α-Ketoglutarate (AKG), Resveratrol, Metformin, and Rapamycin. I won’t go into detail on those now, but I’ll write some more articles about that on my blog soon.
Most of these, however, seem to work mainly as a prevention and not a cure. And while they show a lot of promise in animal models, so far reliable data from humans is scarce. Most of them work through mimicking food scarcity which can also be reached through fasting.
Senolytics
But there are other measures in the pipeline. An interesting idea is the so-called “Senolytics.” Instead of killing themselves as damaged cells normally do, some become senescent. Senescence occurs when cells sense an instability of their chromosomes after having divided a certain number of times or because of high stress (due to their Telomers), so they permanently stop dividing. Senescent cells also secrete signals that lead to inflammation, changing the development of their surrounding cells and the extracellular matrix.
The more senescent cells in an organ, the less vital and functional the organ becomes. Senolytics like Dasatinib and Quercetin are substances that target and remove these senescent cells to rejuvenate the organ. There are ongoing clinical studies on human patients with these substances on several age-related diseases, and they show some promise, but there is still a lot of research to do.
Cellular Reprogramming
The idea that sounds probably most impossible but has the potential to slow the clock and actually reverse aging is cellular reprogramming. Each cell in our body has basically the same genetic information, the same construction plans packed into our DNA organized in chromosomes. But how does a cell in your brain know that it’s not in your foot and has to behave differently? And, even more important, how does a cell know that it’s not supposed to copy itself as often as possible or try to build a new complete clone of you? The answer is epigenetics (mostly). Epigenetics is quite a young field that has made huge progress in the last 15 years. Epigenetics determines which of the genes of a cell’s genome are switched on and switched off by modifying the DNA or proteins associated with the DNA. These bookmarks make a cell behave as it does. They are changed by environmental influences like sunshine, smoking, food, no food, or a thousand other things. Most of these factors and time itself lead to an overall decrease in these bookmarks, although certain areas of the genome also acquire more of them with time. So the idea is to reset these bookmarks to a “younger” state.
Yamanaka Factors
In 2006 a set of four transcription factors (regulators for genes) were identified that can reset a differentiated cell from being part of a certain tissue to a very similar state to that of the cells you find in an embryo. The cells treated with the transcription factors become stem cells and can be reprogrammed into almost any cell type within the body. These transcription factors are called Yamanaka Factors after one of the authors of this study from 2006. Using the Yamanaka Factors, there have been successful reprogramming studies on animals. The aim is to reset the epigenetics of cells to young without dedifferentiating the cells, making the tissues they form fall apart. This technique is currently tested to restore vision in primates after successful tests on mice that have gone blind because of glaucoma. David Sinclair’s group carrying out these experiments expects it to be ready for human clinical trials within this year. If this is successful, it would be a new hope for many blind people and be a proof of concept for rejuvenating a tissue by epigenetic reprogramming.
This is however a very ambitious time line and I dare say it won’t happen. The main reason is that the Yamanaka factors used here are some of the most potent oncogenes. Those are genes responsible for the transformation of a cell into a cancer cell. It is to be expected that therapies working on a thin line between dedifferentiation and cancer will be looked upon with extreme scrutiny by the authorities before being accepted for human trials.
Making and using induced stem-cells from a patients biopsy to heal genetic or other deceases or even target aging, Illustration CC by Manal Hadenfeld, 2020
A possible future application of this could be to treat a patient’s cells outside the body to become stem cells and then inject them to regenerate damaged tissue or to rejuvenate the patient as a whole.
Much is unclear about reversing aging. Many studies in the field show contradicting results, but what would have seemed impossible 20 years ago is rapidly evolving from promising basic research to clinical trials. Currently, you still need some discipline and changes to your lifestyle if you want to increase your lifespan and healthspan. However, the more life and health you win through your life choices, the closer scientists might be to real solutions to all the unpleasant effects of aging and maybe to aging itself.
This post has first been published as a guest post on the blog BoldedScience.com and has since been modified and updated.
Why do we age? What seems like a biological necessity is actually not. Single cell organisms in most cases could be called immortal although the definition is somewhat problematic since for most it is hard to distinguish between mother and daughter, between individual and a population of clones.
There are however also more complex life forms that are not aging as we do. The only really biological immortal animal we know of is the jellyfish Turritopsis dohrnii. It can rejuvenate itself by re-entering the polyp stage and re-emerging as a jellyfish. But there are also mammals that defy our traditional understanding of aging: The naked mole rat.
The naked mole rat is a funny animal through and through. They look like sausages with teeth that could use a dentist. They live underground in insect like states where only the queen is able to breed. Becoming the queen normally means beating the current queen in a bloody usurpation. They have an amazing immune system that basically completely protects them from illness, cancer and infection. And they do not age like we do.
Aging for us is mostly defined by a decrease in physical fitness, the slow loss of function of our bodies after the end of the reproductive phase. It is also an increase in mortality rate (the probability to die within a fixed amount of time). Naked mole rats however show neither an end to reproduction, a decrease in health or fitness nor an increase in mortality with age. That doesn’t mean that mole rats are immortal. On average they become 15 years old but that is mostly because of injuries from fights or predators.
If aging is not mandatory, why is it so common in nature? There are two main theories. The first is that aging is a genetic function that is selected evolutionary since it purges individuals after their reproductive phase which means when it doesn’t affect their evolutionary fitness anymore. However this is already the first problem with this theory. Why should something so consequently be selected in so many? An argument could be that it is an advantage to make room and save resources for your offspring but that’s not really realistic in this case. The selective pressure would be different depending on the scarcity of resources but life spans are not.
The second theory is aging is just an effect of wear and unrepairable damages that accumulate. So is a body just like a car that, no mater how much you put into into repairs at some point it’s not worth it anymore? Obviously this is a factor there are measurable accumulations especially in genomic damages and epigenetic deregulation.
What speaks clearly against pure random damage leading to biological aging, is that it has a clearly distinguishable phenotype. We all are able to tell if a person is old. This seems like there are biological integrators and common pathways activated by different kinds of damage. There are two kinds of those pathways. Those that repair or prevent the damage like stress responses. And those that in case the damage becomes too much and bears the risk of turning the cell into a cancer cell. Those can be programmed cell death in extreme cases but also senescence. Senescence means that cells permanently stop deviding behave differently but stay alive.
This is responsible for many of the observable phenotypes of old age. The more senescent cells an organ contains the older the whole organ behaves.
Back to the main question: Why do we age? The answer is, that we don’t know yet. It seems however that it is a mix of wear and tear that is integrated by molecular responses to the damage and a deregulation that is due to the loss of selective pressure after the end of the reproductive phase.
If you want a deeper dive into the topic from a scientific perspective a good starting point could be the review article by Shufei Song and F. Brad Johnson (2018).
In case you’re not sure how to get scientific publications without paying the horrendous sums many publishers charge for them, stay tuned there will be an article on this topic soon.
