Category: Aging

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.

Transforming Yeast or how to make a GMO

Actual footage of me changing the genes of a hapless yeast, own work

In the last article I ended with a DNA fragment, that I created with a PCR. Now I need to get this DNA into my yeast cells to teach them to age like normal people. This is called transformation. There are several different transformation techniques but most common are elctroporation and chemical transformation. You can spark emotional debates if you ask what is better but the more common and generally a bit easier is the chemical transformation. That is also the method I chose for my aging yeast.

How transformation works mechanistically is not completely understood but the main idea is that you have to overcome the barriers protecting cells from their surroundings. These barriers are the cell wall (for all except animal cells who don’t have one) and the cell membrane. The cell wall’s main responsibility is mechanical stability. You can imagine it like a steel frame that prevents the membrane, which you can imagine like a water bomb, from swelling too much until it bursts. On a molecular level it is a relatively wide net, so it’s not a physical barrier for the DNA molecules. What’s problematic however is that it is electrically charged. DNA is negatively charged and would be repelled from the mostly also negatively charged molecules in the cell wall. This obstacle is overcome by adding positively charged ions that neutralize the charges of the cell wall and allow the DNA to get close to the cell’s membrane.

Schematic structure of a budding yeast cell with a special focus on membrane and cell wall on the right, Giulia Coradello, 2021

But than it has another problem which is the cell membrane. The membrane is a double layer of lipids, fat molecules that have a polar and an uncharged tail. This structure is something like a 2-dimensional liquid. It is relatively elastic and allows uncharged molecules that are not too big to path quite well. Since DNA is huge and strongly charged it normally won’t pass easily. To pass this barrier you have to torture your yeast a bit (*laughs manically in evil scientist). You submit them to a sudden increase of temperature to 42 °C from room temperature. Doesn’t sound too bad but yeast likes it best between 20 and 30 °C above 40 °C starts killing them slowly. This is thought to built differences in pressure between the hot outside and cool inside of the cell which is equalized by the formation of pores in the membrane. Through these pores the DNA can now enter the cell.

To avoid another line of defense of the cells which is enzymes that eat DNA, you also add some carrier DNA, which will be eaten first and so protects the DNA you want to get into the cells. As carrier DNA you normally use Salmon Sperm that you boil and then add to the mix. So if you hear molecular biologists talk about cooking some sperm it’s probably not as weird as it might sound.

After overcoming the defenses you now have a lot of dead cells, many that have taken up nothing or only the carrier DNA but you hopefully also have some that have the DNA that you wanted to introduce into them. What happens with this DNA depends on its form. If it is a closed circle and includes some special signals in the code it will be just kept in the cell and will be copied to the daughter cells when the cell replicates. This is called a plasmid and is good if you just want to introduce genes more transiently without permanently changing the genome. If the DNA (like in my case) is a linear open molecule produced by PCR and has ends that resemble a sequence in the genome of the yeast, it can go into the nucleus of the cell, where the genome is kept. There it can jump in while the cell replicates and integrate permanently into the genome.

Now you have to filter for those cells where this has worked from the rest. You do this with selection. You seed your cells to a medium where only those cells can grow that have taken up your DNA. This is done by adding a marker to the DNA which is a gene that either makes your cells resistant to something that is poisonous to the others or that let’s them live of a food source that the others can’t use. I used cells that normally can’t make Uracil (which is a part of DNA) themselves so they can’t grow on medium where there is no Uracil. They only can grow on this medium if they have taken up my construct which includes a gene that helps them make their own Uracil.

The problem is that this integration is very rare. You start with a huge amount of cells, transform them seed them all to the selection plate and sometimes get nothing, sometimes one or two colonies but sometimes, if you’re lucky, you also get something like 100.

Selection plates of the different transformed strains and a control that was not transformed, own work

To verify that the construct is really integrated where I hoped it would, I now need another PCR. I chose primers that normally produce a short fragment of DNA from the region where I want to integrate my construct. If it is successfully integrated the fragment gets longer which I can see in a gel electrophoresis. And that is what I’m currently doing. I’ll soon give you an update when I’ve finally found my aging yeast.

Teaching Yeast to Age like normal People Part III

Yeast cells growing on a selective plate, own work

In the post “Teaching Yeast to Age Part II” I explained what I tried first to teach my yeast to shorten its Telomers at each cell division. I also told you that it did not work yet. While I have not yet completely given up on Tretrad dissection, I decided to first try another way. It would already be nice to have my fluorescently tagged cells with a knocked out Telomerase but it would be a lot cooler to be able to switch the Telomerase on and off at will.

power on and off switch on wall
Photo by Mikhail Nilov on Pexels.com

The way I’m trying to achieve this is called promoter shuffle. As you might know, Proteins (like the subunit of Telomerase I’m interested in) are encoded in the genome by a fixed code where 3 letters (called a codon) in the DNA (bases) are translated into one letter in the protein (amino acid). This code is well known and understood. All codes for proteins start with the same start codon and end with one of three stop codons. However to really produce this protein in the cell there needs to be more. The gene needs to be flanked by regulatory code that influences if and how strongly the encoded protein is produced. Most important for this is the so called promoter region. This is the code directly before the start codon. It is normally between 50 and a few hundred bases long. Their code is a lot less understood than that for the protein. There are strong and weak promoters, promoters that are always active and promoters that are switched off or on on specific stimuli.

Since I want to make the Telomerase switchable I take the genetic code for one of its subunits and exchange some of the code before its start codon against something else. There are a handful of common switchable promoters in yeast that are well described but most of them have some disadvantages. I identified a relatively newly identified one that I hope will be a good choice.

It is the promoter of the gene DDI2 that is normally almost inactive but increases its activity more than 1000-fold if the cells are incubated with the chemical cyanamide. In the concentration it is used here cyanamide has almost no effect on the yeast cells.

Exchanging code in the yeast genome luckily isn’t too complicated. Yeast quite readily incorporates genetic material that you feed it if a stretch at the beginning and end of the inserted code match the code in the genome. This process is called recombination. Recombination is also the process that makes children unique instead of copies of their parents because it randomly mixes the genetic material of your parents that is passed on to your kids.

So I need a stretch of DNA containing the 500 bases before the start codon of DDI2 with 50 bases on both ends from the area before the start codon of the Telomerase subunit. I did a lot of thinking how to build this myself but it would have been a lot of work. Then I thought about the paper where I first read about the DDI2 promoter. In the nature article “Fine-tuning the expression of target genes using a DDI2 promoter gene switch in budding yeast” Professor Yu Fu of the Chinese Academy of Sciences and colleagues characterized the promoter and explained how they integrated it in genomic locations.

So I just wrote an email to Professor Fu asking if I could get some of the material they used. He actually replied within a day and said that he’d be happy to help me. He sent me the code of the promoter without expecting anything in return. That’s a great thing about the science community. Most people are happy to share and are really nice if you show interest in their work.

So all I have to do now is add the code stretches at the ends that determine the target where I want to integrate it. I’m doing this with a PCR which I’ll explain in my next article.

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.

Teaching Yeast to Age Part I

crop unrecognizable black father disciplining adorable attentive son at home
Photo by Monstera on Pexels.com

In my last article I explained the aim of my project, which is making Telomere length of yeast visible. I also explained the first problem for this: Yeast holds its Telomers constant by a tightly controlled use of Telomerase. That means it doesn’t show Telomere attrition as a sign of aging like our somatic cells do.

This is why I have to teach yeast to age like normal people first. The most obvious strategy to do this is to switch off their Telomerase. In molecular biology you normally do this by removing or destroying the gene or in this case one of the genes that code for Telomerase. We call this a knock-out mutation or just knock-out.

One of the big advantages of yeast as a model organism is, that there are libraries of yeast strains available where you can not only find yeast strains with any protein tagged with Green fluorescent protein (as I explained last time) but also strains with any gene knocked-out. An exception are obviously genes that are so vital that knocking them out makes the yeast in-viable. The genes associated with Telomerase are a bit special in this because yeast with one of them knocked-out is viable but only for a certain number of generations (about 400). One generation under ideal circumstances is about 90 minutes for yeast. So after cultivating them for about 4 weeks their first Telomere become critically short and their cell cycle is stopped permanently. They go into a phase of senescence like our somatic cells at that point.

So basically it would be easy to just thaw one of these knock-out strains from our – 80 °C freezers and I’ve got yeast that ages. The problem however is that I also need a mutation from another library. As I explained in the last article I want to use Telomere associated proteins that are fused with Green fluorescent protein as a marker. So I’ve got a few options: I can take a strain with one mutation and do the other mutation myself. I’ll tell you another time how that works.

Or I can bring both mutations into one cell by mating the two strains. Now I have to explain another cool feature of yeast as a model organism. You might have heard that all the genetic information in your cells is kept in two versions. One from your father and one from your mother. This is called diploid. It has some advantages to have this. The most obvious is that if an important gene is broken on one of your chromosomes there is still a second copy of it from your other parent that can fix it. On the other side in our germline cells (eggs and sperm cells) there is only a single copy of each chromosome. This is made by randomly mixing the the chromosomes you’ve got from your parents. This is also why each egg and each sperm is genetically different. This is called haploid. This form is obviously practical for sexual reproduction because it stops us from collecting more and more copies of the same information in our cells. But it is also great for me as a molecular biologist because if I want to switch off a gene I only have to do so once. There is no second copy that could make trouble.

Now comes the great thing about yeast: It can stably live in a haploid and a diploid form. There is no male or female yeast but there is something similar: The mating types Mat a and Mat alpha. Haploid (only one set of chromosomes) yeast can be either Mat a or Mat alpha and can only mate with the other mating type upon which they produce diploid offspring and stay diploid. Diploid yeast is both Mat a and alpha but not at the same time. It normally changes it’s sex every time it produces offspring. Diploid yeast only mates with diploid and not with haploids even it it currently has the opposite sex. To make diploid yeast haploid again they need to sporulate. Spores are a dormant form of yeast that the produce in bad times. In the lab you create bad times for them by first giving them ample sugar and than suddenly putting them on a very strict diet.

So how does this all help me produce a haploid yeast strain with mutations from two different strains? I first cross the two mutant strains of opposing mating type from which I get a diploid strain with one of the mutations on one set of its chromosomes, the other mutation on the other. Then I bring them to sporulate by setting them on a diet. In the process of sporulation one diploid cell produces four genetically different haploid spores by randomly sharing parts of their chromosomes from each of their two parents. These four spores stay together in one bag (called the Ascus) until times get better and they grow out again and mate. Now if I’m lucky one of these 4 spores has both of the mutations I want in it.

Now the big trick is finding it and this is done by sorting single tiny yeast cells. I’ll show you this crazy technique next time.

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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