Aging and rejuvenation

Steven Clarke

date: 2009-12-17

video: https://www.youtube.com/watch?v=BwsU7nafC_g

LLM-generated summary: In this UCLA Faculty Research Lecture, Steven Clarke elucidates the biochemical battle underlying aging as a dynamic conflict between spontaneous chemical degradation of biomolecules—manifesting as epimerization, isomerization, oxidation, glycation, and other lesions in DNA, small molecules like S-adenosylmethionine (AdoMet) and aconitates, and proteins—and countervailing biological repair mechanisms, including enzymatic epimerases, isomerases, reductases, and methyltransferases such as L-isoaspartyl/D-aspartyl O-methyltransferase (PIMT). Genetic perturbations in model organisms reveal compensatory adaptations via insulin/IGF-1 signaling, adult neurogenesis, proteolysis, and cross-resistance to stresses, underscoring aging not as passive decline but as adaptive resilience, with implications for healthspan extension through signaling modulation, exercise, and phytochemicals rather than isolated gene overexpression.

video description: Aging is warfare between chemistry and biology says Steven G. Clarke, a Distinguished Professor of Chemistry and Biochemistry at UCLA and an authority on the biochemistry of the aging process. Clarke focuses on the fascinating dichotomy between two crucial disciplines, chemistry and biology, and how protein modification can regulate biological function. Series: UCLA Faculty Research Lectures.

Summary

Introduction and Honors

One of the things I've learned in my life is that the biggest honors that one can get are those that come to you at home. On the road, many sins can be covered up, but at home, everybody knows your name. So I'm truly humbled at the honor of this lectureship.

When I walk around the UCLA campus and pass one of the buildings named for a previous faculty research lecturer, it's no longer going to be Hedrick Hall to me. It's going to be Earl Hedrick Hall. It's no longer going to be Campbell Hall to me, but Lily Campbell Hall. And it's no longer going to be Franz Hall, but Shepard Franz Hall.

And I'm particularly proud to have my home department, chemistry and biochemistry, named for William G. Young, the 1947 faculty research lecturer, and my laboratory home in the Molecular Biology Institute, named for Paul D. Boyer, founding director of the MBI in 1962 lecture, and still active in the office across the hall from mine.

So I want to pay particular tribute to the 1960 lecturer, Don Lindsley in psychology and physiology because he was the one that in 1966 hired me fresh out of John Muir High School as a lab helper on an amazing project to understand the neural basis of attention. I worked in a lab in the Brain Research Institute. Our experiments lasted 18 hours, four people. The lab was filled with electrical equipment and wires and as I walk to get a late dinner one night I would go down to the third floor and pass biological chemistry I would see a nice young scientist in a lab coat rattling a few test tubes on a bench and I figured, well, this is for me.

So what I want to talk today about is aging and rejuvenation, chemistry and biochemistry at work.

The Central Feature of Aging: Loss of Function

And the central feature of aging is a slow loss of function. And in Greek mythology, Oedipus certainly knew this when he confronted the half-lion, half-female winged sphinx guarding the gates of Thebes and asking her riddle. Those that couldn't answer the riddle were devoured, but Oedipus knew the loss of function and aging, answered the question correctly, and got the keys to the city.

In more modern times, J. Alfred Prufrock knew this when he was trying to court women when they were only interested in Michelangelo. And he says, "With a bald spot in the middle of my hair, will they say his hair is growing thin? My morning coat, my collar mounted firmly to the chin, my necktie rich and modest, but asserted by a simple pin, they will say, but how his arms and legs are thin."

But probably, I think, the most accurate attribute this loss of aging as Gary Larson, who has the two dogs on the steps, and not only are they letting the mail carrier go by, but the mail carrier is a cat.

So what do we know about the loss of function and aging? And my colleague at the University of Washington, and now UCLA, George Martin, the 2007 Roy Walford Memorial Lecturer at UCLA, has a laundry list. And you can read through this laundry list, but it's basically reduced loss of, reduced heart function, reduced lung function, reduced skeletal muscle function, bone loss, changes in skin, hair loss, sensory losses, losses of neurons, memory losses. And our own Chancellor adds to this list from his work outside of Murphy Hall in the laboratory that in aging, there's disrupted sleep patterns from disrupted biological oscillators.

So, lots of bad news. And sometimes it's bad because in fact not only is it a slow process but sometimes it doesn't seem so slow.

Data on Age-Related Decline and Glimmers of Hope

So if I look here at a measure of the loss of neurons in the brain what I see is there a lot of scatter in this data. But if I look at myself here towards the bottom, if I'm at the top of my game, I'm still at the bottom of a game of UCLA undergraduates.

And then this is actually marathon times. These are record marathon times, not average times, but record marathon times. And this data's a little bit old. It's from 1980, but it plots the best time for runners of a given age. Now, there's three things that you can look at that's not bad news in this graph. The first is the best time is a runner at 31 years old. So at 31, they're besting those 20-year-olds, and so this curve is coming down. They're doing better with age. The other thing is, even though there's the generalized loss in time as one gets older, even the early 50s, the runners are doing as well as teenagers. And most amazing to me is this last point. At 79, at least somebody can still run 26 miles. So perhaps there's hope.

And Deborah Burke at Pomona College gave a wonderful lecture last June, and she pointed out that one of the things in terms of memory and cognition is there is a loss in function in age. And she pointed out that if you test, in this case I think it was Berkeley undergraduates and older people from the community, you read them a passage that perhaps not all of us are familiar with on Mississippian culture some thousand years ago, and you ask them for their recall, the young do better than the old. Now the young still aren't doing very well, but perhaps that's because they're Berkeley undergraduates. But the amazing part of this story is to look at the thing with Berkeley professors. Because not only do all of the professors do better, but actually the senior professors are doing better than the younger professors. And so the question is, is there something that the mind can do by perhaps exercise that actually slows the effects of aging?

But the best data for me was this data from Deborah Burke on actually the effect of age on doing crossword puzzles. Now, you wouldn't catch me wasting my time doing a crossword puzzle, but if I was I would actually be near the top of the game here doing better and better. And so what I take from this is that in fact there is hope.

And I love this picture. This is Muscle Beach Santa Monica in 1948. And I know you're all looking at this glorious example of humankind flying in the air. But I don't want you looking at her. I want you looking at this old geezer here. Because that old geezer, probably five minutes before, was flying in the air just as well. He's just not as photogenic. And so the point is that we actually can do very well in aging.

So my conclusion is that aging is not going to be just a decline without a response, but an active fight of organisms against the loss of function in aging. Not inevitable loss, but a loss that's mitigated by countermeasures.

Dedication and Research Questions

And I have two more slides of this. A year ago last summer we celebrated Paul Boyer's 90th birthday party in the MBI and here's Paul with his friends spanning some generations and the thing that Paul said to me afterwards he said, "Well Steve we'll see you in 10 years at my 100th." So God bless you Paul.

And I'd like actually to dedicate this talk to my mother, Ann Clark, sitting here with her children and grandchildren. She couldn't be here today, but she's still hanging in there. In fact, just yesterday, she managed to get kicked out of hospice for doing too well, for being too healthy. So God bless her.

So my questions for today, then, is on a biochemical and chemical level, what are the mechanisms operating to keep us going against this inevitable decline of aging, and what can we do to slow the decline and enhance the countermeasures.

Hypothesis: Chemistry vs. Biology in Aging

Okay, my hypothesis, a simple one. Battle, war, war between the evil forces of chemistry and the good forces of biology. Chemistry knocks us down, biology brings us back. And chemistry is the bad guy.

Now, okay, what are we made of? We're made of combinations of atoms, carbon, hydrogen, nitrogen, sulfur, and other atoms, and they're held together by electrons, electron sharing. Now, the point is these bonds are not fixed in stone, but they're mutable. Atoms are forming new bonds all the time and breaking old ones all the time.

The problem is, is we make a perfect human, we make perfect molecules, and all of the atoms are in the perfect places. So with the spontaneous changes start, there's nowhere to go but down, okay? There's nothing that can happen but bad news. And this ruination of biology is what we see here, where, in fact, the molecules that are stable are things like water. So if I took a jar of water and let it sit for a million years and came back, I think I'd still see that jar of water. If I take something biological and put it in a jar, even overnight at room temperature or even at body temperature, you don't find the same molecules there. So with the passage of time, there is an inevitable decline.

All right. So now, we're humans. We're tough guys. All right. So I love this sign. When you're starting off on a trail, it gives you such confidence. If you encounter a mountain lion, what to do. But I think it's a great thing for us today because if you see a mountain lion in the wilderness, if attacked, fight back. Okay? Don't take it. Fight back. And that's really my theme for today with aging. There's going to be a mountain lion out there, but it's possible to fight back.

Okay, so then how does the good guy defend itself? And there's three things I want to mention. One is that damaged molecules can certainly be broken down to their constituents and rebuilt, and these pathways are very important. They're well known, but the pathways I want to focus on today are less known, and that their damaged molecules can be repaired. And that these two processes are actually important to cellular turnover, because we can always make new cells, but if you don't break the molecules in the first place and you can keep them going, then you don't have to make new cells. And if you make new cells, you always have the problem on the other side of aging, which is cancer.

DNA Damage and Repair

So what's well appreciated is DNA damage and repair in aging. We have one molecule of DNA in our cells and that molecule is beset by hundreds of spontaneous reactions. Living in water at 37 degrees is mutagenic and those loss of bases can cause mutations. The good thing is our DNA strands have an army of enzymes, an army of proteins. In humans, probably some 200 proteins that spend their lives walking up and down the DNA strand looking for damage. And when they find damage, they fix it. So these things have been known. You have damage occurring. You can't stop the damage, but when it occurs, if you can recognize it with proteins, you can fix it.

What's not so well appreciated, and it's going to be the theme of my lecture today, is that small molecule and protein damage can also be repaired in aging.

Small Molecule Repair: AdoMet Epimerization

So I want to start with an example of non-DNA damage and repair, and I want to start with actually a very simple case, and this is a case of a molecule of ammonia. Now, this molecule of ammonia has a nitrogen atom and three hydrogens. That molecule is sitting there as my hand. Okay, so here are the three hydrogens. Here's the nitrogen. And it's sort of, it's not so sure what it wants to do. And once in a while, it becomes flat. And when it becomes flat, it can flip over to the other side.

All right, now this molecule of ammonia can flip over and you say it makes no difference. But if it was my hands with some asymmetry, if I try to flip it now and put my fingers together, my thumbs now are in the wrong orientation. And if this is a biological molecule where the thumb is expected out here and it's now out here, that's bad news. Okay, so you can think of this as sort of the umbrella in the wind, and what's important is the umbrella in one direction works well, the umbrella in the other direction doesn't work so well.

So the molecule I want to talk about is one of my favorite molecules, S-adenosylmethionine. And except for ATP, so crucial in energy generation and usage, AdoMet is the most widely used cofactor in nature. And in fact, some of you may be actually eating it on a daily basis as a nutraceutical because for reasons that aren't clear to us, consuming about a gram of S-adenosylmethionine each day puts you in a better mood, makes your joints stop aching, and if you've been drinking a lot can help repair your liver.

So this is the molecule, and the molecule is a sulfur atom with three carbons, and these carbons are coming out at you and what this molecule can do is things like transfer this methyl group. This methyl group can be plucked away by nucleophiles the rest of the molecule acting as a leaving group. Or we can take this aminobutyrate group or from decarboxylated AdoMet the aminopropyl group and make things like polyamines like spermidine and spermine.

So the orientation around this sulfur is crucial, and guess what? Chemistry can happen to good molecules. So here's the good molecule where you're looking at the three carbon atoms around the sulfur. For this molecule to work, the methyl group has to be in the back. So just like ammonia flips, this molecule can flip through a planar transition and the methyl group can be in front. And now the enzymes that come to use it bring it on and they say, okay, here it is. Now there's no methyl group here. Where's my methyl group. This molecule now is age damaged and useless.

So how does biology fight back? Well, what it has to do is recognize, in fact, that there's a problem, recognize there's damage. And one of my students, Chris Findeisen, was able to find the enzymes in yeast that recognize this bad guy and convert it to a good guy, methionine, using homocysteine. And in this experiment, he shows the activity of this enzyme with bad epi-AdoMet and with good AdoMet.

So this enzyme sees good AdoMet, it wants nothing to do with it. It says, okay, I checked you out, you're fine, go on your way. But when it sees the bad epi-AdoMet, it reacts it and makes methionine out of it. And when it does that, then, it actually leads to the loss of this product.

Here we show an experiment where we're looking at the concentration in cells of the good and bad forms of AdoMet. In wild-type cells, it's all good, but in cells lacking these enzymes, here's the bad stuff creeping up and becoming almost as concentrated as the good material.

So what happens in this repair pathway then is good molecule goes through aging to be bad molecule. Bad molecule is responded to by biology and is converted into metabolites in the cells that can be used that are good. So the problem basically disappears.

Small Molecule Repair: Aconitate Isomerization

It not the only example. Another example that we found through serendipity in the lab is a detoxification reaction and this is a reaction that occurs in the heart of metabolism. When we teach undergraduates metabolism we teach them the 20 reactions of glycolysis in the citric acid cycle. The students think we're torturing them until we know that there's actually about 3,000 molecules that are really keeping us going, and you're just seeing 20, but these 20 are crucial. These are the reactions by which foodstuffs comes into the body, and we make amino acids, nucleotides for synthesis, and we convert fats and proteins to energy.

And one of the reactions in this cycle is conversion of citric acid to isocitric acid. Now, there's a problem, because this reaction involves an intermediate cis-aconitate, which has a double bond. And there's two types of geometry around double bonds, cis geometry, where the bulky groups are on the same side of the double bond, and trans where they're on different sides. And what happens to the cis-aconitate is it can slowly go to trans-aconitate, and that is bad news for cells because in the reaction of citrate to isocitrate that's so crucial in the citric acid cycle, the cis-aconitate that spontaneously in the aging chemistry goes to trans-aconitate makes a toxic molecule that shuts down the whole cycle.

What saves it is an enzyme that comes along and looks for the trans-aconitate and says you shouldn't be here and converts it to an inactive methyl ester and detoxifies.

Enzymatic Errors and Repair: L-2-Hydroxyglutarate

So you can put lots of blame on chemistry. Spontaneous chemistry does that. But it turns out in aging, biochemistry is not perfect and biochemistry has its own problems. And enzymes can make mistakes. And in September, we had a wonderful lecture at UCLA from Emile Van Schaftingen from Belgium and he told us a story again in the citric acid cycle, but now enzymes that convert malate to oxaloacetate. Again, crucial enzymes in keeping this cycle going.

Now what this enzyme does is convert oxaloacetate to malate, and it does that right about 99.99% of the time. But 0.01% of the time, when it's going to transfer a hydride to this carbonyl group, it uses the wrong molecule. This still has a carbonyl group. Alpha-ketoglutarate looks a lot like it, except it's one more carbon in length. But a disaster for the cell because the product of this reaction is L-2-hydroxyglutarate, and this molecule is toxic.

So it's going fine, but every time it goes wrong, it makes a toxic molecule. The toxic molecule accumulates, and when it does this in humans, we have a number of physiological problems. Difficulty in balance, muscle coordination, intellectual development, seizures, impaired speech.

So, what to do? Well, I think you've got the idea now. Fight back. Bring on repair. And the enzyme that was discovered by Van Schaftingen is an enzyme that takes the damaged molecule and converts it by an enzymatic reaction to a molecule that's already in the citric acid cycle, a molecule we already know about, and takes care of the problem. No muss, no fuss. And so this is humans, there are about 100 humans that actually lack this enzyme that have those symptoms that I talked about.

Protein Damage Mechanisms

Okay, so those are small molecules. Proteins are big molecules, and I show here a small protein, and they have hundreds, thousands, tens of thousands of atoms. And these molecules, because they're so much larger and they have so many more atoms, are that much more sensitive to environmental insults.

Now what are the insults to proteins? And what you learn is that everything that's good for us is bad for us. Oxygen. We can't do without oxygen, but oxygen is an extraordinarily toxic molecule. When plants basically started making oxygen and pouring it out into the environment as a waste product two billion years ago as they discovered photosynthesis, 95% of all organisms died. It was a massive die-off, and only those organisms that learned how to deal with this toxin survived.

Now, glucose is great, a food source that we can't do without, but in fact, it's toxic too. And in fact, water we can't do without, but water is toxic. And what's really toxic of all of this is time itself, because of spontaneous reactions with these compounds.

And so the picture I want you to have is that we have functional native proteins that are going through all of these blue arrows which are different types of damage: oxidative damage, isomerization damage, glycation damage, cis-trans protein isomerization damage, other types of damage to become damaged.

And then what happens to these damaged proteins? One important aspect of biochemistry is many of these are destroyed by proteolysis, taken back to their constituents and rebuilt. What we're learning in these red arrows is that there's a number of repair reactions. And I want to tell you about some of these repair reactions today.

Protein Repair: Methionine Oxidation

The first of these is a repair reaction that was discovered by Nathan Brot and Herb Weissbach in the early 80s. And this does methionine residues. Methionine residues have an Achilles heel, which is the sulfur. That sulfur is subject to oxidation, either forming a sulfoxide or a sulfone. In general, these aren't good in biology. We go from bad to worse. But what they discovered were two enzymes that, if you got to the sulfoxide, could bring it back to methionine. And so if the oxidation of a protein with a methionine residue went to the sulfoxide and these repair enzymes got to it, they could bring it back. Once there's further oxidation to the sulfone, there was nothing left to do and the protein was irreversibly damaged.

Protein Repair: Glycation and Fructosamine

Second form of repair to proteins occurs the spontaneous glycation of proteins, and these are reactions where a protein with a reactive lysine group that is minding its own business wants to be good, comes across a glucose molecule with a reactive carbonyl group and gets together to form a Schiff base and then an Amadori rearranged product of fructosamine.

Now, this product in itself doesn't seem so bad, except then it forms advanced glycation end products. And these guys are bad news. This protein now has a sugar hanging from it that it doesn't want and actually starts making a mess. And what you can think of this as basically, I've got a leech on my body. I've got this sugar molecule that shouldn't be there. And then once it's there, it starts rearranging and doing all of these other chemistry. None of this chemistry we want happening. This is all bad news. And the worst news is it starts cross-linking. So I have a leech here now. It's crossed my arms together. I'm getting more and more uncomfortable.

What can I do about that? And what Van Schaftingen found was that, in fact, you can fight back with another repair reaction. So basically here are the reactions where a protein a nice lonely happy protein gets mixed up with glucose that it doesn't want to do. It forms fructosamine. And what my brother Jim Fraenkel would say, bad. It goes to AGE. But what's good is this reaction of an enzyme that reacts it to phosphorylate this compound. And once it's phosphorylated, actually good spontaneous chemistry can happen, and that leech is removed, the protein is now free, and the products now can go back into metabolism. And so we can reverse this process. But we have to catch it before this bad chemistry happens. So as soon as this comes, we've got to bring it on this pathway. If we do that, we're in good shape. If we let it go to this pathway, we are not in good shape.

Protein Repair: Isoaspartate Formation

So oxygen's bad, glucose is bad, and just living in water has its own problems, and that's why I want to show you here. There are two amino acids in proteins that never should have been put there. Of the 20 amino acids, these two amino acids, asparagine and aspartic acid, should have never been put there because they have a reactive carbonyl, and that reactive carbonyl sets forth a pathway of spontaneous reactions that form these ring-formed reactions and these isomerized and racemized products.

This is bad news for cells. These reactions are happening to us right now. And I was very proud that two students, Phil McFadden and Dave Murray in my laboratory, found by serendipity and some hard work enzymes that look like they specifically recognize this kind of damage. These proteins go up and down other proteins and check them out. If you have asparagine residue and it looks like it's in good shape, fine. If you have aspartic acid residue that's in good shape, fine. But if that asparagine or aspartate has isomerized or racemized, these enzymes recognize it. And it actually can recognize both the racemized form and the isomerized form.

How do you fight back? Well, that enzyme catalyzes the methylation reaction that allows then for repair. And if I'm good, I can hopefully do this with my hands. Here's a nice polypeptide chain with this side chain. What wrong with the side chain is my fingers is a reactive carbonyl group and that waiting to be reacted and there somebody that bad this amino group in the polypeptide chain. These guys are in the same molecule, and all that has to happen is this thing has to wiggle around so these get together and make a succinimide. And when they do that, the succinimide can hydrolyze. Sometimes it hydrolyzes to come back to the aspartic acid. Sometimes it hydrolyzes to make a kink, and that kink is the problem.

As you sit here, your proteins are kinking. Well, what does the enzyme do? It comes down the chain. If it sees this, it passes it by. If it sees the kink, it stops and says, I've got to do something, makes a methyl ester at this free carboxyl, which lets the succinimide form again. If the succinimide then hydrolyzes back to the isoaspartate, it does it again. But the only way out of the cycle is for the succinimide to hydrolyze to the normal form, and basically you have an unkinking enzyme. So as your proteins are kinking, this enzyme is unkinking it.

And so you can either take the version that I gave you with my hands or here's the more chemical version of it.

Summary of Repair Systems and Future Approaches

All right. So this is where we are. Age damage to DNA can be repaired. Age damage to small molecules can be repaired. Age damage to proteins can be repaired. And these are the examples that I gave you from our work and the work of our colleagues.

One huge question. We're made up of something like 23,000 different genes. We probably understand what maybe 5,000 of those genes are doing. We have an additional 18,000 or so genes that we don't know what they're doing. And one of the things that we'd like to ask is how many types of additional damage are there and how many more types of repair are there? And so the approaches to this problem are genomic, proteomic, and bioinformatic to identify new repair systems. And it's been a very exciting process for us.

Genetic Perturbations and Compensatory Mechanisms

Okay, so chemistry's bad, biology's good, and what do we do in this battle to try to tilt the battle towards biology? And what I want to tell you now is a story that actually totally surprised us with the complexity of nature and how strong cells are and how they basically defy many of our attempts to understand them in a simple fashion.

What's new is that in these reactions of damage and repair, cellular signaling reactions are going. People are tattling, they're telling on these reactions. So as damage is occurring and being repaired, other proteins are going around the cell saying, did you know that so-and-so isomerized? Yeah, I heard, but they got repaired.

So what we all look at now is these cellular reactions. And we learned this when we started messing with the genetics. Now, I wasn't trained in genetics. When I was a graduate student, I went to my qualifying exam, and Wally Gilbert, Nobel Prize in Physiology and Medicine, said, Steve, I see you haven't taken genetics. Well, I was sort of a wise guy, and I said, well, you know, I took a line from Peter Mitchell, you know, to tell you the truth, whenever I see DNA or RNA, I turn the page. Well, that didn't do me so well. And I'm paying for it, okay? And I'm paying for it because genetics is actually quite useful.

And in microorganisms, you can do sort of what I call the windshield wiper experiment in a car. You can take a car and you can take off the windshield wipers and you can ask what happens. Now, if it's a dry day, not much. If it's a rainy day, you might see a phenotype. With more complex organisms, what we're finding is you take off the windshield wiper and something else grows back to take care of the problem. And so these questions and answers have almost been more interesting than the original questions.

So we've been looking at this enzyme that recognizes the kinked proteins and repairs them. And what we're saying is, what happens if we underexpress or overexpress these proteins? And we're walking up and down the phylogenetic scale from the bacterium Escherichia coli to the nematode soil worm Caenorhabditis elegans, the fruit fly Drosophila melanogaster to mice. And the short answer, as I mentioned, things are more complicated. There are compensatory changes and links to a major signaling part of the body, insulin signaling, which I previously thought was just controlling blood sugar. Now we know that it has many more roles in controlling cell growth and the resistance to aging stress.

Mouse Knockout Studies: Lifespan, Damage Accumulation, and Behavior

We did this first in mice, and we collaborated with Steven Young lab at the Gladstone. He now a professor in medicine at UCLA. He probably knows more about knocking out mice than anyone in the world. With my student John Lowenson and his student, Ed Kim, they knocked out this methyltransferase. I love to say that, you know, this is one of those experiments that works out so well. You study a process for a long time, and then you actually take away what's so important. And I like to say that these experiments are really difficult to students. And the students say, no, Steve, you know, the technology, you know, get with it, the technology's up there. I say, no, no, psychologically difficult, because what if you actually made a mutation in your favorite protein, and the organism lived just as well? That's what's difficult.

So in our case, we didn't have to worry, because these mice that normally live 700 days have an average lifespan now of 42 days. So the lifespan's been cut in about 20. So what's the phenotype going to be? Well, what I'd hoped was you have an aging phenotype. The mice turn gray, they start tottering around, perhaps say silly things to their children. But what was the phenotype? And the phenotype was they actually died from seizures.

We scratched our head about that because how is that an aging phenotype? And so we thought, well, maybe the brain is a little different. If we can repair the enzyme in the brain by making a transgene, we could look at the other tissues. So John Lowenson and Ed Kim and Steve Young did exactly that, and we were able then to actually look at mice that were older that had the repair in the brain but not in the other tissues.

And so here's mouse age, and here are mice with the damage accumulation that have the repair enzyme. Here's the ones that don't. The repair is going through the ceiling. But wait, what's happened here? At 80 days, the damage stops. It levels off. There's no more accumulation. What's going on? We don't know the full answer, but what it looks like is another system's kicked in. It says, okay, we don't want to see damage above this level. When it gets to here, I don't care anymore about repair. I'm just going to tear that protein down to its amino acids and start over. And in fact, that's where we see the damaged proteins end up as peptides in the urine. And so we have a compensating reaction, a compensating proteolysis. If we can't fix the reaction one way, we'll fix it the other.

How is that occurring? And one thing we realized then was to go back to the brain because it looked like the brain was the most sensitive to the increased level of damaged proteins. Now mice in their cages at UCLA are very well cared for probably more cared for than our undergraduates. But in nature, it's not so easy. Here's a mouse and here's a snake, and one of the things that mice want to do is they want to find food, but they don't want to be eaten.

And so we decided to look at some of these phenotypes, and I had a wonderful undergraduate in the lab, Ryan Vitale, who said, okay, let's put these things through learning tasks. And one of the tasks is called the open field, but it's really just throwing a mouse into a box, into an empty box. And what mice basically do is they explore the outside. They're a little bit afraid of a snake, and this shows the pathway. But a normal mouse, once it sometime goes, it gets brave and says, well, maybe there's a little bit of Reese's Pieces in the middle of the thing here I can get after Halloween. and so it does that.

But look at the knockout mouse. The knockout mouse can't ever get to the center. It just runs around the outside like a crazy one. And so Ryan showed me this data of hyperactivity. So here's the knockout mouse. They're hyperactive. Here's the normal mouse. And I said, okay, Ryan, that's nice for an undergraduate project, but we can't publish that. They have damaged proteins. Of course they're damaged.

He said, well, Steve, how about this? He said, I next put them on a rotor rod. And a rotor rod is like a log rolling contest for mice. And in this rotor rod, what happens is they're put on a rod that's rotating, and you start speeding it up. And the mouse has to walk on the rod at the same speed the rod is rotating, or they fall off. Mice don't like to fall off that rod, and they stay on.

So here's the data he showed me. Here's the knockout. Here's the wild type. And I said, okay, Ryan, so what? You know, they have damaged proteins they don't do as well. And Ryan says, Steve, look at the data, okay? This is the knockout, yes, but this is the speed they're falling off. The wild types are falling off at 25 RPMs. It takes 45 RPMs to cause the knockouts to fall off. They're better at it. These guys have compensated in some way, and they put one foot in front of the other on that rotor rod. They're not going to fall. They're better at the task. So there's some compensation going on. With the damage that comes in, we compensate by proteolysis, and it looks like we compensate by making a better cerebellum.

And in fact, Christine Farrar got a clue to this because she came to me one day and she said, Steve these protein deficient repair deficient mice have big brains. And she showed me this picture. And okay if you're not used to looking at these pictures she said no they don't. She says oh yes they do. And I said no they don't. And so she did the quantification and sure enough they do have big brains. This is the weight. That's about 0.4 grams for a wild-type mouse. And here are the knockouts. They've got brains that are 30%, 40% larger. And it's not water content, and that those differences are throughout the brain.

Why would the brain be larger? Turns out that the big brain phenotype is known in mice that have deficiencies in insulin signaling. Insulin signaling basically is glucose levels are high in the blood. You've had a big meal. You're then your pancreas, the beta islet cells, secrete insulin. The insulin goes in the bloodstream and goes to muscle and adipose tissues and says, hey, hey, hey, there's lots of glucose around. Let's use it. The muscle cells bring in the glucose. The fat cells bring in the glucose. The fat cells make fat out of it. The muscle cells make glycogen out of it, and we're ready for growth.

And in fact, one part of this is that this whole insulin signaling pathway leads to increase in transcription, protein synthesis, cell cycle, and turns off programmed cell death or apoptosis. So it looked like we were turning on this pathway.

Insulin Signaling, Neurogenesis, and Seizures

Now what's interesting is this pathway is shown to be in mice, in worms, and in flies linked to lifespan. And when food is present, what it does is it says, okay, grow. Stimulates cell growth and reproduction with a normal lifespan. Interestingly, when starvation occurs, you have no cell growth, but you also have resistance to environmental stresses and the animals live longer. And this is the whole caloric restriction argument, which is not clear in humans, but certainly in these organisms, if you caloric restrict, you have a tougher organism.

Okay, so we don't know much about brains, so we brought on Carolyn Houser in UCLA Neurobiology, and Carolyn Houser worked with Christine Farrar, and we were able to show that, in fact, a very dramatic activation of the insulin signaling system. And these are slices of the brain that are stained with antibodies. In the top, we've stained them with the non-muscle myosin II protein just to show that the slices are similar. In the bottom, we've sliced them and stained them with antibodies to an activated component of insulin signaling, a phosphorylated form, the AKT kinase. Here's wild type. Here's the knockout. And especially here in the hippocampus and the dentate gyrus, you can see the increase in phosphorylation. So the insulin pathway is turned on in these mice.

Why are we doing this? Questions abound. Do we loosen up apoptotic control to allow new cell divisions and the dilution of damaged proteins? But there's a cost of less resistance to environmental stresses.

So the next surprise, well, at least to this one, is that these pathways are involved in adult neurogenesis. I was taught that once your neurons are formed, and actually I guess it turns out that in teenagers they aren't all formed in the frontal cortex that makes good decisions until 19-21. But after that, there's, in general, no neurogenesis, except in two places. And one of them is the hippocampus. And this insulin signaling pathway actually turns on new neuron formation.

And so Carolyn and Chris wanted to ask, in fact, if that was happening in these mice. Here they're using, they used a number of techniques, but they used antibodies to a doublecortin protein that's involved in microtubules of newly formed cells, and they were able to show that in the mice with the loss of protein repair, there was about a doubling of neurogenesis.

So we're turning on new cell growth. And what was most exciting is this is showing a new dendrite of a neuron in the hippocampus, and this is then the doublecortin, so this shows the new cell. This is the phosphorylated insulin receptor, and you see these little dots, and each of those dots are new synapses that are forming. So basically, the presence of damage there is turning on the system and saying, hey, let's try to do something to get out of this.

But the something to get out of this could be a problem, because you're forming new neurons at the same time the brain's working. And people have attributed this to the same thing as actually why your computer's running to try to put in a new board, okay? It usually doesn't work that way. And maybe the reason that our mice are having seizures is that these new neurons aren't completely incorporated.

And it looks to be that way because the mice don't give up from the seizures. One of the reactions to seizures is a peptide neuropeptide Y. And neuropeptide Y is an anti-seizure peptide. So when mice are having problems with seizures, they make this neuropeptide Y, which acts against them. Chris and Carolyn look for this, and here's wild-type mice with almost none of it, and here is the hippocampus of these protein repair mice where you're putting out tons and tons of neuropeptide Y.

So the point is that mice are trying very hard to survive, and we have compensations to compensations. So we had a compensation to damage during the insulin pathway that caused seizures. The mice then make a compensation of that of neuropeptide Y.

Overexpression Studies in Flies and Worms

Okay, so that's turning it down. Now, the better question might have been, well, listen, if this is so good, why not turn it up? And would this turn down insulin signaling and lead to a longer lifespan? We don't know the answer to this yet in mice. One of my students, Kenan McKay, is working hard on this, but we do know the answer to other systems.

And one of them was a colleague of mine at Boston College, Claire O'Connor, who was looking at fruit flies. and she was overexpressing the repair methyltransferase in fruit flies and she first did this at a normal temperature of growth of fruit flies and there was absolutely no difference. Alright, so that's sort of tough. It's a lot of work, no result.

So what she said is, alright, I'm going to make chemistry work harder. I'm going to up the temperature so that these spontaneous reactions go on at a faster rate and I'm going to see if those little flies can take it. And so basically she put them under heat shock conditions to 29 degrees, and now she saw something very dramatic. Here's the survival curve of the controls. The overexpressed forms lived 40% longer. So something good happened with the overexpression.

And actually Kelly Banfield, Tara Gomez, and Pam Larsen at the University of Texas, did the similar experiment in soil nematode worms, and here there was a 60% increase in lifespan when this protein repair gene was overexpressed. So something good was happening here.

All right. What the hypothesis? Presence of damaged proteins you turn on cell divisions they try to survive. The absence of damage on the other hand with the overexpression turns on the defense mechanisms. These kind of defense mechanisms are good against a whole variety of damages, including oxidative damage. And so we have the possibility of cross-resistance, where basically building up the resistance to one kind of damage builds up the resistance to another kind of damage.

Cross-Resistance to Oxidative Stress

So the experiment is obvious. Let's take a different kind of damage. Let's take oxidative damage and see if there's cross resistance. And so this is the experiment that was recently done by Shilpi Kare in my lab, Tara Gomez, and Carol Lindquist. And what they looked at was the responses of soil nematodes to oxidative stress with and without the repair methyltransferase.

And what they found was very interesting. when wild type cells were given an oxidant like juglone a small molecule found in walnut shells they were just fine but when mutants were given juglone they were very unhappy and it's probably hard for you to see in the back but this is one happy worm and there's a bunch of eggs that are being ready to be laid this is a very unhappy mother she's no longer eating well and her eggs basically are hatching inside of her, and she is going to be a goner soon.

So that's basically what happens when you don't have this cross control.

Conclusions on Organismal Resilience

So what's the conclusion? The conclusion I have is aging organisms are tough. They adapt to adversity very well. Give me your chemical damage. I'll use my biology. I'll fix it one way or the other, but I will do my darndest to survive.

Chemistry to the Rescue

Now, throughout this whole lecture, I probably made my chemistry colleagues mad because I've been saying chemistry is evil, chemistry is bad. But there's hope. And I want to mention briefly some work of Ken Houck in chemistry and biochemistry, of chemistry actually coming to the rescue. And Ken is a master at understanding organic structure and being able to predict structures of transition states.

So Ken can take some of these glycation end products, those cross-linked leeches, and he can say, if there's no enzyme that can fix it, maybe I can make an enzyme that can fix it by understanding what sort of a transition state there should be for the disposal of that and then to engineer a protein that would take care of it. And that's exactly what Ken's doing. He spoke at a recent conference on rejuvenation research in Oxford in September and maybe that will be the rescue. So, yay.

Strategies for Human Healthspan Extension

All right. Last question. Okay, how not just to stay even in this war between chemistry and biology, but to have biology win the battle? how to have this guy in the middle lean a little bit towards the biology side. And so we're talking about human health span now.

And is the solution going to be as easy as overexpressing one or more repair genes? I think the answer is going to be no for the following reason. And the reason is that flies live about 40 days. Worms live about 20 days. Even mice live two years. Okay, even with the fastest early aging diseases, humans are 10 times better than any of those. So in a sense, we've solved all of those aging problems already. We're actually much more highly developed, perhaps to the extent that we can't change anything without making things worse.

And so the depressing part of this, I think, turns out to be insulin signaling. because in the well-fed state, we turn on insulin signaling, cells divide, but they don't worry about damage. They turn off the damage repair, and so this is what I call a short happy life.

Now, on the other hand, you can starve. And if you starve, you turn off this pathway, the cells hunker down, they protect themselves, and now you've got a long, unhappy life. Is there any way around that? And I think what the answer may be, and this is total speculation, is manipulate the signaling pathway. Convince the body that we're starving to turn the stress resistance even when we're well fed.

How do we do this? And there's actually companies actually trying to make drugs that will affect this exact signaling and do this, which may or may not be successful. But at every aging conference I go to at this stage, someone sits up in the audience and says, okay, you know, you have all of these drug regimens, you're trying to do this, you're trying to do that. why don't you just exercise and eat your fruits and vegetables? And that usually quiets the house because in fact in every study, exercise has important advantageous things, not only on muscle but on brain. And there's a whole literature that when you exercise you increase your cognition as well.

So we know that occurs. How is that happening? And in hundreds of studies, we know that consuming more fruits and vegetables actually leads to less cardiovascular disease, less stroke, and a greater health span. So as a biochemist, there's something magic here. And what is the magic? And I think one direction that we'd love to go in is to try to figure out in exercise and in fruits and vegetables and other good foods, what are the components that may be the ones that actually turn the signaling response to be more resistive and to lead to healthspan.

Closing Theme and Acknowledgments

Okay, so the theme here is fight back. When faced with a relentless march of spontaneous degradative chemistry, fight back. The point I hope I've convinced you is that the tools are actually mostly built into the body already. We're already fighting back, and what we want to do is take full advantage of those tools, and if we can cheat a little bit with drugs that may mimic their effect, so much the better.

So I promised a tune to whistle. This is the tune.

One, all biomolecules are inherently unstable, evil, bad chemistry.

Two, successful aging requires active processes to limit the accumulation of these spontaneously damaged molecules. Good biology, and perhaps with Ken Houck, good chemistry.

And finally, the control of the signaling reactions may tip the balance between good and evil.

So I'd like to thank the people who actually did this work. My father used to have a bohemian expression. There's many a mickle to make a muckle. many hands are needed. I've been blessed with wonderful undergraduate students in the lab. The ones in red are the ones presently there, graduate students and advanced researchers and have been feel very happy in my 31 year career to have so many fantastic collaborations outside the lab in the UCLA biomedical community.

And so I just want to end with a little Bob Dylan: May your hands always be busy. May your feet always be swift. May you have a firm foundation when the winds are changing shift. May your heart always be joyful. May your song always be sung. And may you stay with repair enzymes forever young. Thank you.

Q&A

Q: (Layperson on SAMe and aspartame): I'm a totally lay person. I couldn't possibly follow all the details. but I recognize two brand names in the course of your talk. And one was the, I think it's pronounced SAMe, S-A-M-E. And the other is aspartame. And I wondered whether the one was good, the other was bad, or what is the relation of what you talked about to those two?

A: Okay. Actually, the sweetener aspartame, I didn't talk about. I was talking about aspartic acid, which has... But I'm wondering if you've had the same taste since it's the same. Actually, what's interesting about aspartame is that it's a dipeptide, and it's subject to the same kind of reactions that go on in the cells. And we've actually looked at this and asked whether damaged forms of aspartame can build up. And I think they actually do because soft drinks that have aspartame in them, if you drink them after letting the can sit for a few months, they're no longer sweet. And so I think that same chemistry is going.

The SAMe, S-adenosylmethionine, I think is a much more exciting story. So I'm someone that uses S-adenosylmethionine in these enzymatic reactions. So actually, in three of those repair reactions, S-adenosylmethionine is used. Fantastic cofactor. So I love it. So when these reports came out that eating it was good for you, that's fantastic. Here's my favorite molecule. You eat it it's good for you. And it's amazing how good it is for you. And as a mood stabilizer it as good as any of the tricyclic antidepressants. The only problem is it about 100 times more expensive.

But for psychiatrists, when they have patients that come in and say, I want something natural, I don't want to take a drug, they say, hey, take adenosylmethionine, you already have it in you. So what is it doing? We don't know. But what I think is happening is completely the opposite chemistry. And I think what happens is it doesn't get into cells, it actually spontaneously breaks down itself, and the breakdown products get into cells and actually inhibit methylation reactions. And it turns out that methylation reactions are involved in inflammation, and we're too good at inflammation. And so basically, because of that, we tune it down and we're healthier. And so we can stop the kinds of inflammation that leads to cirrhosis of the liver, that leads to osteoarthritis of joints. The effect on mood, who knows how that's going, but I think it might be the same way of actually turning down these pathways.

Q: (Male Speaker 1 on cancer): Early in your talk, you talked a little bit about cancer. And so when you talk about having these natural repair mechanisms go on, how does that relate to what cancer does?

A: So, you know, there's a dichotomy of cancer and aging. If I had a seesaw, I could use that same slide for aging on one side and cancer on the other. Because if you want to live forever, all you need to do is sort of what a redwood tree or a bristlecone pine does and let your cells continue dividing. And so if your cells continue dividing, all of this damage you can dilute out. But the cost of that is losing the control of those cells and leading to a tumor.

And so, in fact, if you want to really be very careful about cancer, you can stop cancer entirely, but in doing that you'll age fast. If you want to age a long time, you're going to have cancer come with you. And the best example I have of that is gray hair. I know a little bit about that. And there's some arguments about why hair grays, but I think the best explanation I've seen is that we have stem cells along the hair root that regenerate themselves and then form melanocytes at the base of the hair follicle and lead to the color in the hair.

So these stem cells can become anything and so they do a good job of keeping color in the hair. But they have a problem. The stem cell has a problem that because it can be anything it easy for that cell to become a cancer cell. And so when you turn gray actually it might be sort of a beneficial reaction because what you're doing is you're saying let's lose those melanocyte stem cells because if we lose them, maybe we won't have melanomas that form. And so that's sort of the toss-up we have.

In the brain, the same type of a toss-up. When I talked about neurogenesis, if we could make new neurons, why aren't we doing that all the time? Why do we wait for damage? So we make new neurons in Alzheimer's disease. We make new neurons when you get a massive hit to the head. And why don't we do it all the time? And I think the reason is that when you use these stem cell measures, there's danger. And the danger is those cells are going to lose control and become cancerous.

And so that's why I emphasize that, you know, those models where you can make new cells are great, but it's tempered by the fact that you can lose control. And so my thought is the body does all of these efforts to basically stop that damage before you even have to make a new cell. So if you can keep the old cell happy, then you never have to come to that problem. If you can't, you may have to go to that and make that decision of cancer versus aging.

Insights

• Core Intuition: Aging as Chemical-Biological Warfare. Aging arises from thermodynamic instability of biomolecules in aqueous environments at 37°C, driving spontaneous damage (e.g., epimerization via planar inversion in sulfonium centers like AdoMet, cis-trans isomerization in aconitates, succinimide-mediated isoAsp/D-Asp formation in Asn/Asp residues, oxidation of Met, glycation of Lys); biology counters via targeted repair enzymes that recognize damage stereospecifically, often using AdoMet as cofactor, rather than wholesale proteolysis or turnover, minimizing cancer risk from excessive proliferation.

• Mechanistic Insight: Repair Cycles and Specificity. PIMT exemplifies repair: methylates α-carboxyl of isoAsp, destabilizing to succinimide intermediate, enabling hydrolysis back to L-Asp (probabilistic ~50% success per cycle); methionine sulfoxide reductases use thioredoxin for stereospecific reduction; fructosamine-3-kinase phosphorylates for spontaneous deglycation; epimerases for AdoMet use homocysteine to regenerate Met, preventing cofactor depletion.

• Trick: Damage Surveillance and Thresholds. Enzymes patrol polymers (DNA, proteins) for lesions, ignoring native forms; knockouts reveal damage thresholds triggering compensatory proteolysis (e.g., elevated urinary peptides) or signaling cascades.

• Main Concept: Compensatory Networks and Insulin/IGF-1 Signaling. Loss of repair (e.g., PIMT KO mice) activates hyperinsulinemia, driving hippocampal neurogenesis (doublecortin+, p-IR markers), cerebellar compensation (rotarod superiority), but causing seizures (countered by NPY); overexpression enhances stress resistance (heat shock in flies +40% lifespan, worms +60%). Cross-resistance: isoAsp repair boosts oxidative tolerance (juglone survival in C. elegans). Cancer-aging tradeoff: proliferation dilutes damage but risks oncogenesis; repair preserves post-mitotic cells.

• Healthspan Strategy: Mimic Starvation Signaling. Caloric restriction/off insulin pathway upregulates repair/stress resistance; exercise/fruits-vegetables likely activate via phytochemicals modulating TOR/insulin pathways without actual nutrient deprivation.

Transcription errors?

• "8-O-MET": Likely "epi-AdoMet" or "(R,S)-AdoMet" (the inverted epimer at sulfur); "bad 8-O-MET/good 8-O-MET" contextually refers to sulfonium epimers.
• "Sami, S-A-M-E": Clearly SAMe (S-adenosylmethionine).
• "Aminobuteral": "Aminobutyrate" (from AdoMet).
• "Isospital": "Isoaspartate".
• "Non-muscle actin": Likely "non-muscle myosin II" from context (brain staining control).
• Minor: "Adenosylmethionine" sometimes "acidnosomethionine" (speech slur); "PCMT" implied as PIMT (protein L-isoaspartyl methyltransferase); ambiguous phenotypes (e.g., "big brains" quantified as 30-40% heavier) assumed accurate. No chemistry ambiguities beyond standard terms (e.g., cis-aconitate confirmed). Overall, high fidelity post-correction using biochemical knowledge.