Category: Overview

How to Stop the Clock

grayscale photo of man thinking in front of analog wall clock
Photo by Brett Sayles on Pexels.com

Can we slow or even reverse ageing?

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 Hallmarks of Aging CC from Rebelo-Marques 2018

What is aging?

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.

What’s a PCR and how does it work?

In the last article I ended having a DNA template thanks to Prof. Yu Fu and I had a DNA sequence of the region where I want to integrate my DNA construct. So to now I need to copy a piece of the DNA plasmid (ring) that I have and add a stretch of DNA sequence (~ 50 bases) that are complementary to to the point in the yeast genome where I want to integrate that peace. I can do both in one step with a technique called Polymerase Chain Reaction or PCR.

A PCR is a method to produce DNA without using living cells to do it. It is something like a molecular copy machine. The main ingredients for a PCR are an enzyme called DNA-polymerase, which does the copying, dNTPs, which are the building blocks of DNA, the template DNA from which I want to copy and primers. Primers are small DNA peaces (normally about 20-25 bases long) that define the DNA stretch I want to copy. The forward primer (the start signal) is complementary to the sequence where I want to start the copy and the reverse primer is identical to the sequence where I want to stop the copy.

Schematic drawing of a complete PCR cycle
Schematic mechanism of PCR. CC BY-SA 4.0

The picture above is from the Wikipedia article on PCR that also explains it really well in detail. As you see the first step of a PCR is making the DNA accessible for the copying by heating it until the typical double-strand structure splits up into two single strands. For this you heat it to something around 95 °C. Then you cool it down to the annealing temperature which depends on your primers but is normally a bit above 50 °C. To start the DNA-polymerase you now heat it again to something around 72 °C. This sounds like a very high temperature for an enzyme and it is. The trick is that the polymerase comes from the bacterium Thermophilus Aquaticus (or others) that lives around hydrothermal vents that spew water, smoke, acid and other stuff at temperatures of more than 100 °C. So their enzymes are optimized to work in extremely hot and unpleasant conditions.

Now you repeat these three steps over and over again and with each repeat the number of your DNA constructs doubles. This means you have an exponential growth from 1 to 2 to 4 to 8… to a bit more than a million after 20 cycles to a bit more than a billion after 30 cycles. So you create a lot of identical DNA in quite a short time (normally 1 to 4 hours).

The machine that adjusts the temperature in a programmed manner is called a Thermocycler and looks quite unspectacular.

A typical laboratory Thermocycler made by Eppendorf, own work

This explains how I copy the stretch of DNA from the plasmid but I also wanted to add some sequence to the ends. That’s actually quite easy. I just add the sequence to the primers because they are the beginning and the end of my newly created DNA constructs. Now you might ask: And how do you get these primers? Well like most things today. I order them online. There are companies specialized in producing custom DNA. Basically I could also save myself the whole work and just order my whole construct online but that is still quite expensive. While my exceptionally long primers (primer + sequence are about 60 bases long) cost something between 20 and 50 $, my whole 2500 bases long construct would cost about 1.000 $ to order. That is still quite a difference but it gets cheaper and in a few years biologists will probably order a lot more DNA than they create using molecular techniques like PCR and cloning.

Now that I have my PCR product I want to verify if it is the right one. The easiest way to do that is to put it on a gel that lets short DNA pass quicker than long DNA. Than you add voltage and the negatively charged DNA wanders towards the positive electrode and splits up by size. If you add a marker that includes DNA pieces of known size and a compound that makes DNA visible under UV-light (ethidium bromide) you can see what size the fragments that you produced have. That normally gives you a good idea if the experiment ran as expected.

I did all of that and on the first try the result was no visible DNA at all. Than I changed a few parameters and tried again and saw a very thin band where I expected it and a few others where they shouldn’t be. So I changed some more parameters and on the third try I got this wonderful picture of a gel with a strong band at exactly the position where I expected it.

This made me quite happy but now I had to do something with it. I wanted to put this DNA into a yeast cell and make it integrate into the genome. This is called transformation and is quite tricky as well. I’ll explain that the next time.

Teaching Yeast to Age Part II

In my last post I explained how to get a bag of 4 yeast spores of which one hopefully has the two mutations I want in my new yeast strain. Now comes the really tricky part: You have to find this spore.

Yeast cells under 1000x magnification (DIC), own work

Before I explain the method you have to get an idea on how small one yeast cell is. The average yeast cell is about 3 to 4 micrometers. The smallest distance between two dots to still see them as two with the naked eye is 0.1 mm which is about 300 times a yeast cell. A human hair is on average between 0.06 an 0.08 mm thick, which is 20 times a yeast cell. If you look at the picture above, depicting yeast cells magnified 1000x, the whole frame is about the thickness of a hair.

OK, so they are really small and not visible with the naked eye but we want to find a single spore (that is again smaller than a normal cell) and identify it. The method we use for that is called micromanipulation. A micromanipulator is basically a microscope with a stage (on which you fix the plate with your cells) that can be moved in tiny increments. And it has a lever with a very thin glas needle fixed to it that through some mechanics can also be moved in a very small scale.

My lab has quite a cool and modern micromipulator that has a joystick to move the stage and a computer driving you to positions on a virtual grid on your plate where you can put single cells.
What you now do is you look through a patch on your plate where you’ve put some of your cells. Once you’ve found a bag with 4 spores (which is called a Tetrad) you move the tiny needle really close to it. A moment before the needle would actually touch the plate a meniscus of water forms between the needle tip and the plate that scoops up the Tetrad and hopefully nothing else. Then you move to an empty position on the grid and, by moving and shaking the needle while it almost touches the surface, you try to deposit one of those 4 spores. Then you go to the next spot and do that again. So in the end you get a plate full of 4 spots in a row where you deposited single spores. You can see how it works in the following video.

Me showing how a Tetrad dissection works.

After finishing the sorting I put the plate to 30 °C for one or two days to give the cells time to grow. The result looks like this:

Dissection plate with colonies on it, own work

As you see not all cells I’ve laid out have grown. That is quite normal because not all spores are viable after the procedure. On the left side was the reservoir, where I’ve put a lot of cells to look for Tetrads. I’ve cut that off with a clean scalpel so the cells do not overgrow the rest of the plate.

In the next step now I have to identify which of these cells are really derived from spores (and are haploid) and which of them are actually just normal diploid cells that accidentally lay around with 3 buddies and looked like Tetrads. To do this you use another nice feature of yeast. Wild yeast can live of a lt of things and can make everything it needs (like amino-acids and bases for DNA) semselves. The labstrains you normally use however have some genes they need for this knocked-out. This means to let them grow you need to add some amino-acids and other things to the medium. Now the trick is, that the genetic traits that I want to breed together here are marked with the genetic code that the laboratory strain needs to make these amio-acids themselves. That means I take this plate and stamp it over to a plate where the medium is missing two special amino-acids, only those cells can grow that have both genetic traits that I need. This is called selection.

The problem here is that the diploid cells I mistook for Tetrads also have both markers and can also grow. Here however the statistic can help. Since I always put all 4 cells that were together in one row and in a Tetrad only one of those 4 can have both of the genetic markers, I now have to check my selection plate for rows where only one of the 4 cells has grown. Another test that I can do afterwards is to see if the cells of such a colony mate with other haploid cells of either mating type. Diploid cells won’t do that.

Original plate on the right, selection plate on the left, own work

The results look relatively promising but further analysis showed that this actually hasn’t worked. However this is an important part of science: Things don’t work. After trying this a couple of times I decided to first try another way to teach my yeast cells to age that is a bit more elegant and hopefully works better: I will put a switch on the gene for Telomerase so I can turn it on and off as I want. I’ll explain how this works the next time.

What are Telomeres?

To explain what I’m doing I’ll first explain what Telomeres are and why their length is relatively important.

Telomeres Structure and function from Kupiec, 2014

Telomeres are solving a problem that came up when bacteria became eucariots (plants, mushrooms and animals). At this point they stopped storing their genetic information on DNA-rings and instead developed linear chromosomes. This brings some structural advantages for bigger chromosomes but has a huge disadvantage. Each time a cell divides (which most cells do a lot) it has to replicate it’s genome so each daughter can have a copy. This is done by enzymes called DNA-polymerases. The process only works in one direction on the DNA-string and doesn’t start at the beginning.

For a circular genome this is no problem but for a linear genome this means the loss of a few bases (the building blocks of DNA) at each devision. For a while this is not a huge problem if you don’t write important stuff at the beginning (or end) of the chromosome. And that is already the first function of Telomeres. Aside from mechanically stabilizing the chromosome ends, they are a peace of genetic code at the ends of chromosomes that do not contain vital information.

This is already enough for many of our somatic cells (those cells in the body that form most of our body) they only divide a certain number of times until a tissue (like a muscle or your brain) is formed and then just work and do not or only rarely divide.

Only cells that need to divide unlimited or often (germ line or stem cells but also unicellular life) need another trick. This trick is called Telomerase and earned it’s discoverers a Nobel Prize. Telomerase is an enzyme complex that uses an RNA-template to elongate Telomeres and so completely solve the so called End-Replication-Problem.

The fact that most somatic cells are not using this trick and have telomerase switched off has an interesting implication. It makes Telomeres something like a cellular clock of aging. They limit the number of times a cell can divide and rejuvenate a tissue before it goes into a state called senescence. The more cells of an organ are in this state, the less likely the whole organ gets to repair itself and to function correctly.

Another type of cell that needs to replicate a lot is cancer. This is why about 4 out of 5 cancer cells have Telomerase switched on.

The full picture of the connection between Telomeres, aging and cancer are quite complicated and still under discussion but it is quite clear that influencing Telomere length has quite some potential to treat cancer on one side and influence aging on the other.

If you’re interested in a good overview on the current state of knowledge on the connection between aging and Telomeres there’s a quite new review article from Vaiserman et al., 2021.

If you want further information on Telomers and cancer I’d recommend the article of Hiyama et al., 2003 about Telomerase as tumor marker or Graham et al., 2017 about Telomeres and prostrate cancer.

For an overview on Telomeres as a target for drug discovery see the nature article of Bearss et al., 2000.

A short story about aging

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.

Turritopsis dohrnii medusa
Turritopsis dohrnii medusa (CC by Bachware)

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.

Naked Mole Rat
Naked mole rat in Zoo (CC by Roman Klementschitz, Wien)

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.

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