Immortal Reactions

By Simon Quellen Field

In the last few years, research has exploded in the field of longevity. We now have an understanding of most of the mechanisms of aging, giving us a handle on slowing the process, and a pathway to reversing it.

Explore the reasons we age, why we age more slowly than most other animals, what goes wrong, and what to do about it. Delve into the latest science and what is in store for the very near future.

• Language: English
• Publisher: Simon Quellen Field
• Release date: Feb 3, 2023
• ISBN: 9798215882214

Book preview

What Causes Aging?

Why do some organisms live longer than others? Many flies live only a few weeks. The record lifespan of a mouse is about five years. A bowhead whale might live 200 years (Keane, 2015), and a North Atlantic quahog clam can live 500 years (Austad, 2010). Deep sea corals may live as long as 4,000 years.

Animals that have fewer predators tend to live longer. It is thought that animals in risky environments don’t evolve mechanisms that spend energy on repair. They favor spending energy on reproduction. Large animals tend to live longer than small animals presumably because they are less prone to predation. Naked mole rats live 30 years or more because they stay underground. Bats live 30 years or more because they can fly away from predators. Turtles and tortoises can live for 160 years because their shells protect them from predation.

What goes wrong so quickly that the fly can only live a few weeks?

There are many things that can damage an organism at the cellular level, which is where aging occurs.

We know that a byproduct of oxygen metabolism is what is known as reactive oxygen species (ROS). These are chemicals that can damage tissues (but they are also important signaling molecules, as we will discuss later). We also know that every time a cell divides to make new cells, there is a chance that the DNA strand might break, or there might be an error in the production of new DNA, so that nonsense information is encoded and the gene no longer functions, or functions less well. Environmental damage can also occur. Ultraviolet light can cause DNA damage in the skin, causing cancer. Viruses can damage cells and DNA. Bacteria can produce toxins, and the organism can eat poisons that damage cells and DNA. An organism’s own immune system cells can produce peroxides to kill bacteria, and these can damage nearby cells.

Organisms have mechanisms to prevent or undo this damage. Reactive oxygen species can be mopped up with antioxidants, either those produced by the organism (such as coenzyme Q-10 or glutathione) or those found in food (such as the colorful compounds found in many fruits and vegetables). Breaks in DNA are repaired by a number of different mechanisms. How much energy an organism spends on such damage control and repair has a big effect on lifespan.

Enzymes are proteins made by an organism to cause specific chemical reactions. Three of the main enzymes used to mop up reactive oxygen species are superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). Because enzymes are proteins, long chains of amino acids folded into complex shapes, they are expensive to produce. The energy and raw materials for making damage control enzymes could be used instead for reproduction, feeding, escaping predators, or growth. Each organism evolves different trade-offs between growth, reproduction, and repair. If an animal lives in a high-risk environment, spending energy on repair makes less evolutionary sense than spending that energy on reproduction. They aren’t likely to live long enough for the repairs to pay for themselves.

Long-lived animals have better control over reactive oxygen species, and better DNA repair mechanisms. There is a clear inverse correlation between the number of somatic mutations and lifespan across all species (Cagan, 2022) indicating more efficient DNA damage repair. There is also a clear inverse correlation between the size of an organism and the number of somatic mutations, indicating better control of the mutation rate in larger animals. But eventually, even a bowhead whale dies of old age. What is happening?

If DNA damage in a cell escapes the repair mechanism, the genetic code can be changed. These changes in the genetic code of non-reproductive cells are called somatic mutations. Since they are not in reproductive cells, they are not passed on to offspring, but cells with these changes can reproduce, creating colonies of cells in tissues that operate differently from the rest of the cells around them. Cancers are one type of somatic mutation, but other, less deadly mutations can gradually build up, making tissues less able to perform their necessary functions.

But DNA is not the only information storage mechanism available to cells. Much of the information in a cell is about which genes to turn on or off. This is what allows every cell to have the same DNA, and yet become different tissues in a growing embryo. This extra information is called epigenetic information, and is stored in markers on the DNA (called methyl groups) or markers on the proteins that help to package the DNA. Those protein markers determine whether the DNA is packed up tightly so that it cannot be read, or whether the DNA is open to the enzymes that copy the information and use it to make proteins.

Proteins called sirtuins are involved in adding and removing this epigenetic information, but also in repairing breaks in the DNA. If there is a lot of DNA damage (caused, say, by smoking cigarettes), these sirtuins proteins spend their time repairing the DNA, and are not available to maintain the epigenetic information. This leads to cells losing the differentiation that makes them do their specific job in the tissue they are a part of. The information that this cell is a liver cell, or a heart cell is lost. Over time, this loss of information causes the effects we call aging.

Cells can often detect damage, and commit suicide or stop reproducing, to avoid the spread of non-functioning tissue. So-called senescent cells are cells that have stopped reproducing, but do not die, living on to making inflammatory molecules (cytokines) that attract immune cells and open capillaries to get more blood flowing to the site of the injury. The inflammation those molecules produce can damage nearby cells, and even cause more of them to become senescent. As we age, we collect more and more of these undead zombie cells, leading to chronic inflammation.

Inevitably, some of the damage is going to affect the same mechanisms that are involved in defense and repair. Over time, the body’s ability to fend off reactive oxygen species damage and repair DNA will be reduced, leading to increasing amounts of damage as time goes on.

What are the odds?

In 1825, Benjamin Gompertz (Gompertz, 1825) looked at mortality statistics and showed that the chances of dying in any particular year increase exponentially with age. The odds of dying double about every eight to nine years after about age 30. Graphing the data from the U.S. Social Security Administration (Administration, 2019) for the year 2019 gives us a chart that looks like this:

In order to see the first part of the chart better, it helps to zoom in closer:

There seems to be a sweet spot just before puberty, at around 11 years old.

The steep drop in the curve at the beginning is a reflection of congenital birth defects, which often cause death soon after birth, but sometimes can allow survival for several years before proving fatal.

The Gompertz graphs can be interpreted as a constant chance of damage occurring, accompanied by repair mechanisms that themselves collect damage with time. If we could maintain throughout life the level of damage control we had when we were pre-teens, the graph would remain flat instead of getting steeper every year.

We can look at what is going wrong with our repair machinery, and examine ways to improve it.

What is going wrong?

In a paper published in 2013, Carlos López-Otín listed nine of what he termed hallmarks of aging (López-Otín, 2013). The first hallmark was genomic instability, the accumulation of genetic damage throughout life.

Related to this is the second hallmark of aging, the shortening of telomeres. Telomeres are the ends of our chromosomes. They are made of DNA, but exist for structural reasons instead of making proteins. The enzymes that copy DNA when a cell reproduces don’t work at the ends of the chromosome. A special enzyme, called telomerase, is needed to make the ends of the chromosome. But most non-stem cells in the body do not make telomerase, or make very little of it. Each time a cell reproduces, the telomeres get a little bit shorter. The purpose of telomeres is to prevent the DNA repair mechanisms from thinking the end of the chromosome is a break in the DNA. When the telomeres are too short, the cell stops reproducing, and in some cases commits suicide, so that chromosomes are not joined together by DNA repair.

The third hallmark is also in the genome. The epigenome is the set of molecular additions to the DNA or the proteins that package the DNA. These additions (or marks) control which genes are expressed, and by how much. As we age, this information gets scrambled, as it is not as well repaired as the DNA coding itself.

The fourth hallmark of aging is the loss of protein quality control in the cells. Proteins have to be folded properly in the first place, and recycled properly when they are damaged. If the mechanisms that deal with these are not working properly, damaged and misfolded proteins can accumulate in the cell, causing loss of function. As we age, these processes decline.

The fifth hallmark of aging is the loss of regulation of nutrient sensing. One of the earliest successes (in 1934 and later in 1939) in prolonging lifespan (McCay, 1934) was the recognition that restricting calories makes organisms from yeast to worms to flies to mice to monkeys (Colman, 2009) live longer and resist the diseases of aging. Limiting calories shifts the organism from growth and reproduction into DNA repair and protein maintenance (Garinis, 2008). Growth hormones are suppressed. Likewise, when growth hormones are suppressed through other means (as in genetically modified organisms or through drugs) the animals live longer and stay healthier longer.

There are four nutrient sensing pathways that affect longevity (Houtkooper, 2010). The insulin and Insulin-like Growth Factor system (IIS) senses glucose levels. The mTOR (mechanistic or mammalian Target of Rapamycin) detects levels of amino acids (the building blocks of protein). The adenosine monophosphate kinase system (AMPK) senses low energy levels, and sirtuins sense low energy states by detecting high NAD+ levels. Each of these systems will be discussed in more detail later.

The sixth hallmark is mitochondrial dysfunction. As we age, the mitochondria in our cells accumulate damage that does not get repaired. Cells repair mitochondria through a form of autophagy called mitophagy.

Autophagy (from the roots for self and eating) is where cells do housekeeping, sweeping up damaged and misfolded proteins and organelles and recycling them to make new ones. Mitophagy is the recycling of damaged mitochondria.

But unless mitophagy is stimulated by something like exercise, caloric restriction, or molecules like spermidine, metformin, or resveratrol, the damaged mitochondria simply accumulate.

Cellular senescence is the seventh hallmark of aging. Senescent cells are cells that have detected damage, and stop replicating so that they do not become cancerous. But they also start emitting inflammatory signals to alert the immune system to the damage. This can cause the chronic inflammation seen in older organisms, and it can cause nearby cells to become senescent themselves, leading to more damage.

Senescent cells are important for embryogenesis and wound healing, but if they accumulate when and where they shouldn’t, they can cause problems. If these senescent cells were cleared by the immune system and new cells created to take their place, all would be well. But things can go wrong with the clearance, and with the ability to replace damaged cells with new ones.

Which brings us to the eighth hallmark of aging: stem cell exhaustion. The same things that cause aging in other cells in the body affect stem cells and their partially differentiated version, progenitor cells. Damage accumulates that is not properly repaired, telomeres shorten faster than the stem cells can lengthen them with telomerase, mitochondria malfunction, damaged proteins accumulate, etc. If stem cells are called upon to proliferate excessively (to replace damaged cells in tissues) the stem cell niche may become exhausted and unable to provide the needed cells.

The ninth and final hallmark of aging is altered intercellular communication. The most obvious form of this is the increase in chronic inflammation that accompanies aging. This is often referred to as inflammaging (Salminen, 2012).

Senescent cells cause inflammation, but so do cells that have not done their autophagy housekeeping, and damaged mitochondria. Worse yet, the inflammatory cytokine molecules that alert the immune system also shut down autophagy, in a vicious circle. Inflammation is involved in most of the diseases of aging: atherosclerosis (Tabas, 2010), diabetes (Barzilai, 2012), obesity, and failing stem cells (Doles, 2012). At the same time, the adaptive immune system declines, so that pathogens, cancer cells, and senescent cells aren’t cleared properly (López-Otín, 2013).

Underneath the hallmarks

So now that we have a descriptive view of what is going on with aging, we can look into the mechanisms of aging.

Sirtuins

We begin with the sirtuins. These are seven genes (SIRT1 through SIRT7) that have many roles in the body. The major one we will discuss is DNA repair, but these genes also activate proteins that are not directly involved with repair, and they are responsible for opening up the proteins that chaperone DNA so that it can be read and transcribed. This last function is where sirtuins get their name: Silent Information Regulator. Genes are silenced when the histone proteins that the DNA wraps around packs them tightly, so they can’t be read. The SIRT genes code for proteins that remove a molecule called an acyl group from the protein, allowing it to be more flexible and open up.

In the image above, you can see the DNA double helix wrapping around a cluster of eight proteins. These proteins are the histones. The DNA strand wraps around almost twice (about 1.7 times).

The rest of the DNA then attaches to another histone and DNA loop, and then another. This is part of the packaging of DNA that allows our cells to cram six feet of DNA into tiny packets (chromosomes) that fit inside cells that are a millionth of a meter in diameter.

Some parts of the DNA strand never open up to be transcribed into proteins. The centromere, in the middle of a chromosome, and the telomeres at each end of the chromosome, are structural. The centromere works to allow the chromosome to replicate itself, and the telomeres act as a stop signal for the DNA repair mechanisms. Without that stop signal, the DNA repair process would mistake the end of the chromosome for a break in the chromosome, and try to fix it by joining two chromosomes together.

Every cell that has DNA has all of the same genes. What makes a liver cell different from a brain cell or a heart cell is which genes are turned off (silenced). We start out as a single fertilized cell, which then begins to divide into a multitude of cells. These cells gradually differentiate into various organs by turning various genes on and off. The proteins that do this are the sirtuin enzymes (Rodriguez, 2013), along with three other enzyme groups.

Sirtuins perform many different functions. One of them is to react to cellular stress, coordinating the response to stress. This is why they are important in preventing cancer, performing DNA repair, and managing inflammation, autophagy, and apoptosis (programmed cell death) (Rodriguez, 2013).

As we age, the activity of sirtuins decreases. All of those functions the sirtuins perform no longer get done as much, or as well. DNA repair declines, cancer prevention declines, inflammation increases, and autophagy (Lee, 2008) no longer cleans up cells as well.

Sirtuin enzymes need a molecule called nicotinamide adenine dinucleotide (abbreviated NAD) to work. NAD is thus called a coenzyme (the sirtuin enzyme only works when NAD is present). When this molecule is in short supply, sirtuins can’t function. And NAD levels in the body decline with age. We’ll see why later.

NAD is such an important molecule that it makes sense to know a little more about it.

We know from the name it is a dinucleotide. That just means it is two nucleotides joined together. A nucleotide is a molecule made up of a sugar (such as ribose), a phosphate group, and in this case either nicotinamide or adenine. Adenine is one of the bases in DNA and RNA. Nicotinamide is one form of the vitamin B3. We can look at the parts separately. Here is nicotinamide:

The blue nitrogen atom on the right with the two white hydrogen atoms attached is called an amine group.

An amine is what you get when you react ammonia (a nitrogen with three hydrogens) with an acid. The acid in this case is nicotinic acid. The part that makes it an acid is the red oxygen double-bonded to the gray carbon.

Adenine looks like this:

It also has an amine group up at the top left.

Ribose (the sugar we need to make our NAD) looks like this:

It is a five-carbon sugar, in this case, in the form of a ring.

A phosphate group is made of phosphorus and four oxygens:

When we put together the base (nicotinamide or adenine) with the ribose and a phosphate, we get a nucleotide. When we join the two nucleotides by their phosphate ends, we get nicotinamide adenine dinucleotide:

A much simpler little molecule is acetic acid, the molecule that makes vinegar taste sour:

The right side of the molecule (a gray carbon, two red oxygens, and a white hydrogen) is what make it an acid (something that can easily donate a hydrogen to something). When the hydrogen is gone, what remains is called an acetyl group:

When sirtuins have enough NAD, they can use it to add an acetyl group to a protein. If that protein is an enzyme, adding an acetyl group can either activate it (the usual case) or deactivate it, depending on the enzyme. But to do this, the sirtuin needs to break the NAD into nicotinamide and the rest of the molecule, which is O-acetyl-ADP-ribose. So sirtuins consume NAD.

Other things also consume NAD, such as the DNA repair enzyme PARP1 (poly ADP-ribose polymerase 1). Inflammation also chews up NAD using an enzyme called CD38 to make molecules that stimulate the immune system. If there is chronic inflammation, the CD38 lowers NAD levels so much that DNA repair suffers, and the sirtuins can’t do their other job of maintaining gene silencing. Cells malfunction, and can go senescent, causing yet more inflammation, in a vicious cycle. Nutrients such as apigenin and luteolin can inhibit CD38, which is the body’s biggest consumer of NAD.

NAD is also important for maintaining the daily circadian rhythms in the body. As we age, and NAD levels fall, the circadian clock begins to fail (Levine D. C., 2020), resulting in disturbed sleep, and altered sleep patterns.

To make the interplay of all of these factors more apparent, we can construct a network of longevity inputs and outputs:

Inputs are in violet, beneficial effects are in green, and red nodes are those harmful to longevity in the long run. Green arrows indicate increase, and red Ts indicate inhibition or decrease. NR is nicotinamide riboside, NMN is nicotinamide mononucleotide, NAD+/NADH is the ratio of oxidized NAD to reduced NAD (more on that later), PARP1 is a DNA repair enzyme, and CD38 is the protein that converts NAD into inflammation signaling molecules.

This network of interconnected molecules and pathways is the heart of our story, and we will gradually grow the network as we look into more aspects of aging. But keep an eye on the violet circles. Those are the inputs we can affect to slow down the aging process.

Our first three hallmarks of aging all have to do with DNA, and sirtuins and NAD are essential to proper DNA functioning and repair. The next hallmark is protein quality control, and for that we need to look at autophagy.

Autophagy

Autophagy (self-eating) is how cells clean themselves up. There are three mechanisms cells use: microautophagy, macroautophagy, and chaperone-mediated autophagy (abbreviated CMA) (Parzych, 2014). While they act in different ways, all three target proteins or organelles (like mitochondria) and break them down for recycling.

In macroautophagy, inside the cell a membrane (called a phagosome) forms around misfolded proteins, malfunctioning mitochondria, and other cellular debris or organelles that are no longer needed.

When this membrane completely surrounds the target debris, it is called an autophagosome. The signaling protein ULK1 is required for the formation of the autophagosome, and inhibition of ULK1 is one important way that autophagy is controlled.

This then merges with an organelle called a lysosome, which contains enzymes and acids that break down the debris into component parts that can be reused. The resulting organelle is called an autolysosome (a lysosome for autophagy).

The autolysosome uses acids and enzymes called hydrolases to break up the contents into small pieces which can permeate through the membrane back into the rest of the cell (the cytosol) through pores in the lysosome.

In microautophagy, the lysosome itself does the engulfing, without needing a phagosome.

In chaperone-mediated autophagy, a specialized protein called a chaperone recycles certain types of soluble proteins by attaching to them and unfolding them into strings that can feed into a special pore in the lysosome. This specialized form of autophagy only exists in mammals (Kaushik, 2011).

Chaperone-mediated autophagy is selective, unlike the other two forms. Only proteins with a particular marker (about 30% of all cellular proteins (Alfaro, 2019)) are shuttled through a special membrane receptor (called LAMP-2A for lysosome-associated membrane protein type 2A) on the lysosome for disassembly. The targeted proteins have a five amino acid tag in their sequence that allows a chaperone protein to attach to them (Cuervo, 2010).

When food is scarce, cells use autophagy to scavenge for the molecules they need in order to function. When an organism like yeast is grown in a solution without enough sugar, the yeast cells live quite a bit longer. When mice are fed a diet that has 30% to 40% fewer calories than they would like, they also live longer. This caloric restriction path to longevity was one of the first to be discovered, and increased autophagy is one of the ways lifespans are increased.

Intermittent fasting (eating only during a small window in the day, or not eating for a day or two in the week) can stimulate macroautophagy and microautophagy. Macroautophagy is activated in the first hours of fasting, reaching a maximum at six hours before gradually subsiding. Longer fasts will stimulate chaperone-mediated autophagy as well.