#09 - David Sabatini, M.D., Ph.D.: rapamycin and the discovery of mTOR — the nexus of aging and longevity?

Foreign. Hey everyone. Welcome to the Peter Attia Drive. I'm your host, Peter Attia. The Drive is a result of my hunger for optimizing performance, health, longevity, critical thinking, along with a few other obsessions along the way. I've spent the last several years working with some of the most successful, top performing individuals in the world, and this podcast is my attempt to synthesize what I've learned along the way to help you live a higher quality, more fulfilling life.

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Find more information on today's episode and other topics@peterattiamd.com. In this podcast, I'll be speaking with my close friend and amazing scientist, David Sabatini. David's a professor of biology and a member of the Whitehead Institute at mit. He's also an investigator at the Howard Hughes Medical Institute and a senior member of the Broad Institute, along with a bunch of other accolades that would take too long to get into here. This podcast was actually recorded initially as part of an interview series I was doing for research around my book, and this was recorded in August of 2017. Maybe at some point we'll even just put the video up, as this was actually done as a video interview with David, along with a number of his amazing postdocs, and certainly some of those will probably make their way into the podcast as well. Now in this episode we talk about his amazing journey in science and the work and stuff that he's done around MTOR and Rapamycin. And if you've been following the blog and or paying attention attention to stuff that I'm interested in, you'll know that MTOR and Rapamycin sit kind of at the heart of it. Now, about four years ago, David and I were having lunch one day and.

It was kind of the first time.

That he ever really told me the full story of his work as a graduate student at Hopkins where he was part of the MD PhD program.

And I was just, you know, I.

Remember sitting there taking notes on a napkin and thinking, God, this is such an incredible story of science. And I remember thinking, God, you know, one day we have to have this discussion again, but such that most people can actually hear it besides just me. So part of what we discuss on this podcast is actually that journey and how As a young PhD newbie grad student, David methodically went after a problem that really wasn't even deemed particularly interesting at the time, which was to basically figure out how this thing called rapamycin actually worked and of course through the process ended up being the first person to identify this mechanistic target of rapamycin in mammalian cells. Now, stuff that I found really interesting in this podcast is that David points out that he's, from an academic standpoint, kind of an unusual bird, in that.

He'S one of the few people who.

Has carried his work from graduate school into his career. And that's actually pretty unusual. He's incredibly thoughtful. And some of you may have already heard a podcast that David, myself, and Nav Chandel, another good friend, who will also be on the podcast, recorded back with Tim Ferriss on Easter island back in the fall of 2016. We'll link to that here as well. And obviously, the reason we went to Easter island was as sort of a pilgrimage based on the discovery of the bacteria that ultimately led to rapamycin, a bacteria by the name of Streptomyces hydrocophagus. The interest I have in mtor, of course, has to do with its central role in nutrient sensing, and, of course, I believe, and many believe, its central role in longevity. So if you are interested in longevity, if you're interested in fasting, if you're interested in rapamycin, you're really going to want to listen to this podcast because David is effectively. I don't think there really is a person on the planet, and I'm saying that without trying to be hyperbolic, but I don't think there's anybody on the planet who knows more about rapamycin and MTOR than David Sabatini. And if you like this podcast, please make sure to check out the one that's going to be out soon with Matt Kaeberlin, which will take this discussion to another level as well, looking at Matt's work in dogs. So without further delay, here's my discussion and conversation with David Sabatini.

Well, David, thank you so for making time to sit down today and talk about what is potentially mutually our favorite topic of discussion. Before we jump into it, though, maybe for people who don't know you, can you tell us a little bit about how you got here and what it is you do specifically?

Sure, sure. So thank you, Peter, for coming and for visiting and both of you, and for wanting to talk to me. So, I am a biologist. I'm a professor of biology at mit, and I'm also a member of the Whitehead Institute, which is where we are today. And I receive a lot of funding from the Howard Hughes Medical Institute, which is a key charity that works with biomedical researchers. I have studied this protein that I'm glad you like a lot. It's My favorite protein called the MTOR protein, which is the protein through which this drug rapamycin, which gets quite a bit of attention now acts. And I basically worked on that from the earliest point. We discovered that when I was a student. And so my career is as an MD, PhD. Never really following the clinical track though, and staying on the research side and finishing that and actually coming to the Whitehead in a program that is quite interesting and very unique at the time, which is that you could start your own lab after graduate school. So I did that and I eventually joined the faculty here and now I've moved up the academic ranks. And to some extent I'm a little bit strange from an academic point of view because I've continued to work on, not exclusively, but to a large extent, what I started in my graduate school. Most people, as you know, do something in graduate school, they do something in their postdoc and then they sort of morph along the way. I've kind of stuck with this MTOR protein and in many ways I was very lucky because we were there at the beginning and it turned out to be such an exciting thing to work on.

So let's go back to the beginning a little bit. You were an MD, PhD student at Hopkins and after a couple of years of doing pre clinical stuff, you pick a lab.

Exactly right. So you do two years of medical school and then you pick a lab. And I was very fortunate to be taken by Solomon Snyder, who was at the time the head of neuroscience. He had a very big lab, lots of MD PhDs, a lot of MD PhDs wanted to go to his lab. And I was lucky that he let me go. And Saul is a really interesting man. He still has actually a really prominent lab at Hopkins. In fact, the department is now named after him. And he was that person who had a lot of varied interests. So he was a neuroscientist, but he was also a psychiatrist and he was also a pharmacologist. So he really loved small molecules and he liked particularly potent small molecules, I.e. small molecules that act at low doses.

How do we define in pharmacology a small molecule? What's the cutoff point?

You know, Some people say 1,000 Daltons, which rapamycin is about that to a large extent. It's sort of a non peptide also. So it's not a piece of a protein in many cases it's not a natural molecule, although in our case it's made by micronormous, but it's not natural to our body. So probably thousands of dons and so he had these set of interests. And when I went to his lab, I was actually really interested in neuroscience. So I'd had some classes in which I was sort of fascinated by some neuro questions. But when I got to his lab, I actually never did anything on neuroscience. And I often told the story that the most influential scientific discussion I probably ever had is when I went to talk to Saul. And basically, as a student, you need to pick a project. This is something that it is quite challenging. And I see with my own students, they really get quite apprehensive about their project. So I went to talk to Saul and I went to his office and I only met with Saul like, I don't know, maybe five or six times during my PhD. So this was like a big deal. And so I went to talk to him and he basically said, david, we work on the brain. And I thought that was great because I wanted to do neuroscience. But then he didn't say anything else, so that was it. And so I remember leaving his office really anxious because basically, like, I didn't have a project. But I realize now in retrospect, what he did is he actually was giving me complete freedom to do what I wanted to do. And that was, as I said, probably the most important thing anyone's ever done for me because it really forced me to come up with my own project and I think was a key sort of foundation in becoming the scientist I have. And it's something that I try to foster amongst my own people. So anyways, I was in his lab and I didn't have a project. And at the time they were actually working with this other drug called FK506, which is A. An immune suppressant, immunosuppressants used clinically still mechanism of action. Although structurally it's very different than cyclosporine, it actually mechanistically works on the same target, which is calcineurin. And at the time, this is before we had a lot of the tools we have now, like rnai or crispr. And so you needed controls. So if you had a drug, what you tended to use was another drug that kind of looked like it but didn't do the same thing. And so their control was rapamycin. And when I started reading it, rapamycin.

And this is what year this would.

Have been in probably late 91 to 92. And it was clear to me that this molecule in many ways was much more interesting than FK506. And as you very well know, this had come from Wyeth Ayers, the pharmaceutical company by Soren Sehgal who championed it. And there was a number of papers which at the time were actually a few papers were largely abstracts from meetings that showed that it had antifungal effects, immunosuppressive effects, anti cancer effects. So it seemed like an interesting molecule. I had just come from medical school, we'd learned about immunosuppressants like cyclosporine, which at the time were really just coming on and were really seen as miracle drugs.

Your Lab's interest in FK506 was not its immune suppressive properties, but its calcineurin inhibition.

Exactly. Because calcineurin, as the name implies, there's a lot of. In the brain. And so in Saul's lab they're basically studying the modulation of calcineurin in the brain using FK506 as a tool. And they were looking actually at excitotoxicity in the brain, things that at the end didn't lead, I think in the directions they wanted to, but they were using it as a pharmacology, as a probe, basically. And so I basically decided why not try to work on rapamycin. And so that's what I did. And so we.

Which was just a control that nobody particularly cared for.

Yeah, I mean there were people in the world that were interested in.

But in your lab.

In our lab, yeah.

People in the lab.

No one was studying rapamycin. We had this great advantage though is that you couldn't buy rapamycin at the time. So rapamycin was a compound that Wyeth was developing clinically. It wasn't clinically available, you couldn't buy it. But Saul, being a very prominent scientist and having this interest in pharmacology, had actually written Suren Sehgal and actually he had sent us probably without any of the legalese that happens now. Now if you try to get a molecule out of pharmaceutical company, the amount of paperwork and red tape is huge. Basically it sent us a very significant amount of rapamycin, which I remember when it did start to be sold, which was incredibly expensive, I kind of back calculated the street value. Yeah, it was like millions of dollars.

Wow.

Now of course it didn't really have that value, but theoretically it was sort of millions of dollars. Which incidentally, that tube followed me all the way here and then.

Do you still have the original tube?

No, at some point it was lost. It disappeared at some point. But anyway, so we actually had it, which was cool. So we could actually do experiments with it. And so I went on to try to understand how it worked. And eventually we purified this protein that at the time we actually called RAFT one was the original name we gave it. And eventually it was called.

And Raft 1 stood for, it was.

Rapamycin and FKBTP Target 1. And the reason that we. And also Stuart Shriver, who was at Harvard and when he was working on this, he was also at Harvard, also identified mtor, basically at the same time he called it frap, which was fkbp, rapamycin associated protein. And both of us were trying to accentuate the point that rapamycin acts with a CO receptor. This protein, fkbp, from that point of view, it's a very unique kind of drug where it doesn't directly bind to a protein target, but rather it first binds to one target and now that drug receptor complex has a new surface on it which now in this case interacts with mtor. And we were really trying to get that point across eventually, but independently.

Independently, we realized we were both working on this.

Yeah, I had no idea that in fact the only point where I found out they were working on it was once our paper had been accepted. I got, and this was pre. Lots of email, Internet. We got a fax from a journalist saying he was writing an article on our paper and another paper from Stuart Shriver and actually sent us Stuart's paper, which we thought was really unethical at the time. And so we actually at that point contacted Stewart and said, hey, we got your paper, you should know we're working on this too, and here's our paper. So yeah, I didn't know at all. And in many ways I was very naive, right? I was in this lab. Saul basically let us do whatever we wanted to. We had this drug. Unbeknownst to Saul, I started working on this thing. And Stuart had had a history of FK506 mechanism action. So it was a logical progression to what he was doing. Saul was not. It was funny. He came from a world where people looked for the receptors for drugs. So if you look at his history, he'd really looked for receptors for drugs for small molecules, including the endorphins, for example, that I worked with. But he wasn't big on cloning, what we call cloning a gene, which is where you get the DNA sequence. He almost thought you didn't need to do that. Once you purified it, you could study the protein. So I was one of the few first people there to actually clone a cdna, as we call it, in his lab. So it Was a fun time because it was clear that we'd gotten this protein.

But you did this in a very short period of time because your paper which was in cell was 1994.

94, yeah. I worked like crazy. Really like crazy. So. And that lab in general worked like crazy. We were, it was very common to be there until one in the morning and then I would usually show up at 7, 8 in the morning. You know, we would sleep in the lab a lot. And once, you know, once things started to go so we were purifying, I purified out of the rat, out of rat brains. And so we killed hundreds of rats to do this. And my friends would help me kill them, take the brains out. There's a method in biology to visualize proteins called a silver stain, which is a very sensitive way of seeing a protein. And the first silver stain I did where I actually saw sort of a glimpse of MTOR on this method, I remember that really clearly because at that point I knew I could do it.

How did you know it was MTOR that you were looking at?

Well, I mean I had all the controls and there was this band on what we call a gel that showed up just in the right place.

I see.

And so I was like, okay, there is a protein here that has all the properties that I want.

And at that time, what properties did you know? You didn't know its size, did you?

Didn't know its size? Well, we know it bound to fkbp, rapamycin.

But you didn't know that that exclusively bound to it, did you?

We didn't, but we knew that it could be competed by FDA 506 based on some competition type experiments. And so we had done that. So there was these features. It was mostly the specificity that it required rapamycin to bind to fkbp. And that was crystal clear. In the early experiment when we had fkap by itself, there was no band on the gel. And when we added rapamycin there clearly was. And when we added FK file 6, it clearly went away. And so we knew that that thing had all the right properties. But I remember very strongly feeling okay. And at the time, now we have very, very sensitive methods to sequence proteins, largely mass spectrometry there we didn't. And so from what I saw in that gel to actually figuring out what its sequence was, I knew it was hard, but I knew it could happen. That was like a very powerful feeling.

It was the existence principle.

Exactly. So I knew the thing like kind of the enemy existed and I could get it but then going from that initial glimpse on a gel to then having enough to actually sequence it, that's what took hundreds of rats to actually get to enough that I could purify it. And eventually we collaborated with this guy called Paul Temps at a Memorial Sloan Kettering in New York. And he was able to sequence enough of the protein that then through a whole variety of molecular biology tricks, we were able to clone it. And it was a really huge cdna, which basically is the length of the DNA sequence that encodes it. It was very big, sort of in the 8, 9 kilobases, which is very hard to work with, particularly at the time. And I got very, very lucky in the sense that I did a bunch of tricks and I got the whole thing at once, which is also was kind of unheard of.

How did you do that?

Yeah, so back in the time, what people would do is they would get pieces and then they would sequence them and they would, with overlapping, they would overlap and stitch them together. But what I did when I screened what we called libraries at the time for these pieces, I would get some pretty big pieces, but I knew when I would sequence it, I knew that the front of the protein was missing. Like I was missing it and I couldn't get it. It would never, I could never get beyond a certain point of the protection protein. And so then what I did, which you know, really turned out to be like incredibly lucky. So what we would do is we would screen libraries of phages. And so this was basically people would take cdna, complementary DNA from rat brain and they would clone it into these bacterial virus called phages. And so now every little CDNA was in a virus and you'd have hundreds of millions of this library and you would plate it out on these plates and the phage would make little plaques and then you would screen those plaques. And so you'd have dozens of these plates, each with thousands and thousands of these little dots on them. And so what I decided to do is that I would screen this library with a piece that I knew was as far towards one end and as far toward the other end. And so I screen it with both. And I looked for plaques that, that hybridized both. And in fact, when I first did it, I got nothing. It was really disappointing. I got plaques, I had one piece, I had plaques, I had another piece, but I didn't have any plaques the same. And then what I realized, and this was really key, I realized that the so called full length CDNA was so big that it was going to make the phage replicate slowly because basically their genome was so much bigger now that to replicate it would take longer and.

On what order of times?

It was probably two to three times more that it would take.

So you could have been missing it?

I could have been missing it because the plaque that would have this would be incredibly small. And so what I did is I went back and redid it and now I let the plaques grow longer and I rescreened it. And in fact I got one plaque. There's a tiny, tiny little plaque that hybridized with both probes. And when I looked at what was in there, it turned out to be the complete full length cdna, which was amazing because it was unheard of that these libraries would give you something like 9,000 base pairs. But it was, when I see cornstate, it was literally the intact thing from one end to the other. So I got very lucky because that would have been pretty hard to assemble at the time.

So you knew at the time. Had Michael Hall's work in yeast been published yet?

It had been published sometime during this period of time, but we didn't.

But you didn't know anything? You didn't know even what the yeast form of this?

No, no. When we started, we were enjoying it. And in fact, when we first started getting sequenced, there was no sequence out there. And the yeast protein, really only the kinase domain is concerned. And so most of the peptide sequences that we had that Paul temps had sequenced for us, we didn't know at all where they were. Right. And so these are kind of fun things that used to happen in the past. You used to collaborate with a person who did protein sequencing and they would give you back a series of peptide sequences, but you didn't know what order they went in. So let's say he gave you back. I think Paul gave me. Paul was amazing because he would give you, let's say 15 peptide sequences. He'd say, look, your protein, these 15 peptides exist in your protein with or without overlap.

In those peptides, no overlap.

These are short.

Mathematically, it's impossible to by chance figure out. Like you need more clues to figure out the order because it's combinatorily impossible.

Yeah, you have no idea what the order is. And so what you end up doing is kind of a cool thing. And Paul was really cool because he would actually, in the sequence of the peptides, he also had uncertainty. Sometimes he'd say this amino acid plus or minus could be this or could be that. And he would tell you what other each and it turned out he was so good that when I actually figured out the sequence, every one that he said it could be this or that, he was right, his prediction. But so what you would do is you'd have these peptide sequences and what you could do now is design. We know the, obviously the code, the amino acid code, so we can predict what the DNA sequence would encode. But as you know, the DNA sequence is degenerate, right? So one peptide sequence can be encoded at the DNA level.

You don't know what the extrons and.

Introns look like around the world.

You don't know anything. Right. But each peptide could be encoded potentially by thousands of oligonucleotides and you don't know the order of the peptides. What you would do is you would make a degenerate pool of oligonucleotides that had thousands of different ones and you'd make them in both orientations. And now you do PCR between them in all combinations and you would find which ones worked and that would define the order of the peptides.

And this is before you had real time pcr.

Yeah, real time PCR usually used for quantitation, but we had pcr. And so we would take these oligo libraries and we'd mix and match them all combinations in all orientations. And if you got a band, it means that you got that you had basically figured out. And then you could take those fragments and go and screen the libraries. And so it's funny because now with my students, when we discover a new protein, all you do is you look up in the database because we have the whole genome sequence. I always tell my students that my paper which was the discovery of mtor, which at the time, to be fair, we did not realize how important MTOR would be. My paper basically is like figure 1 above of their papers because my whole paper is about discovering the proteins, sequencing it, all this kind of stuff.

Was that paper effectively your PhD?

That was my PhD.

So you went back to finish a couple years of med school, obviously decided not going to do a residency. I'm going to become a full time scientist. And then you basically have been at MIT since or affiliated with MIT since.

Saul's lab was big and I was very independent. So the people said, why don't you do one of these fellows positions where you can start your own lab. And at the time there's only three. There was the Whitehead one, there was one at Carnegie Institute, which is in Baltimore, and there was one at cold Spring Harbor, N.Y. and I applied to all of them, and I got accepted pretty quickly, although after I graduated to Cold Spring harbor and to Carnegie, But I didn't hear anything from Whitehead. Like, nothing and only, like, basically once I graduated and I was kind of unemployed at that point. I was technically a postdoc in Saul's lab, but I hadn't taken, like, the boards, which Hopkins. You know, Hopkins didn't make you take the boards, the medical boards, to graduate, which was a nice thing. My mother was like, you're gonna starve. You don't have a job. You can't do residency now because you didn't apply. You didn't take the boards. And then I got a call from Whitehead actually inviting me to interview, and I did. And then it took again a lot of time to, like, hear back. And I remember they called me and said, look, we're gonna offer you a position, but you need to understand you will never, ever stay here as a faculty member, ever. I was like, okay. I realized I was applying for this Whitehead fellow position, not a faculty position. But then eventually I came, and eventually I did stay. And when I look actually at history, they do keep about a third of the people who come through, but they give you this sort of speech that.

You will never, ever see, set the expectation.

And incidentally, many of the people named David have stayed. So it's actually a good thing to be named David. Actually, our current director was a Whitehead fellow. His name is David. One of the other faculty members. His name is David. So I didn't know at the time, but now I realize that David was a big advantage.

So how has your work evolved? I mean, you came here in the late 90s, right?

In the late 90s, exactly.

Rapamycin would go on to be approved by the FDA in 1999 as a frontline treatment as part of the double or triple cocktail for patients.

As rapamune.

Right, right, as rapamune, along with often prednisone, cyclosporine, or mmf. So now you're here, and, I mean, we're going to get into much more detail, but effectively, you've never looked back. You've never really left this space.

I got here, and I was incredibly naive. I realized at this point how I thought, you know, I knew a lot. I thought I knew how to run a lab. I had been very independent on my own, but that doesn't mean that I was sort of independent from, like, running a lab, you know, behind the scenes. In Saul's lab, obviously, it was the entire finances. I had written grants and things, entire finances, organization. But there was a lot of like I could be independent me, but then a lab is a different thing. And so that was a hard transition to run, even though it was a small lab to run a lab. And it was clear that at that time I felt that this field had kind of plateaued. There had been the discovery of mtor, but we weren't getting very far. People were using rapamycin to look at lots of different things. And mtor by implication through rapamycin was being connected a lot to the. But one of the things that was obvious to me and I think to others as well, was that MTOR had to act with partner proteins. And so we set about trying to identify what we now know are these mtor containing complexes mtorc1 and mtorc2, mtor complex 1 and 2. But that was. It was really hard. We failed for years and it was again one of this field has had a series of just like little things that until you figure them out, you make no progress. And so we would purify MTOR and we'd look for other proteins. We would continue to work with poltemps and we just wouldn't find anything.

To be clear, you knew that you had discovered the gene for tor, you suspected that this thing exists in different complexes.

And I already knew that there was other proteins because when I was doing the MTOR original purification, the way that I was following MTOR was with kind of a funky cross linking assay where I was crosslinking a radioactive FKBP to the putyer of target and there was always two bands on the gels. There was a protein MTOR which I eventually purified, but there was a smaller one which I could never get either because it was just low abundance. I couldn't detect, I don't know what. But that little protein which at the time I called Raft 2 actually basically remained unidentified. So I knew that there was something.

So basically the first version Of MTOR complex 1 was TOR and the version of MTOR complex 2 was RAF2.

No, no, no. That protein actually now that we found it turns out to be in both complexes.

Oh, I see.

But what I knew was that there was an associated protein with mtor. I knew from. I didn't know what its identity was, but it was very clear on all my experiments that there was a small MTOR is very big, it's around 300 kilo Daltons, which is a big protein. This was a little protein, it was around 30, so it's about 10 times smaller so from a technical point of view, it's about 10 times harder to get because there's about 10 times less peptides in that protein. So I failed to get it. So when I got here to the whitehead, I knew there was another protein to find. And we kept trying to go after this protein and others. We knew it had to work. You know, it's a really big protein. Big proteins work with friends. And it turned out this is again, these little things. It turned out that the detergent. So when you work with mammalian cells, you have to lyse them, you have to break open their membranes. You typically use a detergent. Turns out the detergent that we were using, which is by far the most common detergent that every lab in the world use, breaks apart these complexes just by bad luck. Just bad luck. And I had a postdoc, his name was Doss Sorbozov, who figured this out. And he found this other detergent called chaps that kept them together. When you think back your career and you're like, well, what are like these key inflection points? His discovery of that detergent was key because once we did that, we purified all the interacting proteins. And that eventually led to MTORC1 and MTORC2, eventually led to all the proteins associated with those. Basically, that was the key to all the biochemistry. There was like several years of nothing. Then he found that, and then everything has sort of. From that point on, we've sort of marched along in figuring out all the components of this pathway. We still don't know why things are sensitive to Triton. We don't know why they're incentives of the detergent. But it's the kind of happenstance of science that I guess makes it interesting.

So when, roughly by year, where are we when we have a we, meaning the world, as a result of your discoveries in the lab, where are we when we sort of know that now we have MTOR Complex 1 around Raptor, MTOR Complex 2 around Richter. This is.

This is around 2002. Right? So we were doing that around 2001, published around 2000. It's in that range. It's in the early 2000s. Although, as I said, we knew there was complexes even back in 94.

And at this point in time, your thought was these two complexes control what, or sense what, or are important for what?

Right. So it was very clear early on that MTORC one was doing most of the things that we had connected before to mtor. So, you know, we'd had rapamycin and so Rapamycin, in a way, had allowed us to know a lot about MTORC1. We now realize than otherwise we would have known, because we didn't have really genetics, we didn't have easy ways of modulating mtor, but we had rapamycin. And so there was a body of knowledge acquired by many different investigators about what was so called downstream of mtor. What did MTOR do? We had some ideas. It was a growth regulator. It regulated translation, it regulated autophagy, it regulates many, many metabolic pathways, regulates cell size. We knew that largely through the use of rapamycin. And so now when we discovered MTORC1, which the first part we discovered was this protein called raptor, we now could go and say, well, does raptor matter for all those things? And it turned out it did. So it was very clear that MTORC1 must be doing the things that we ascribe to rapamycin. Mtorok2, therefore, remained very mysterious for a long period of time because it wasn't doing those other things. And only later did we find it was actually part of the PI3 kinase pathway and a regulated AKT. And that clarified lots of things. And in many ways, MTORC2, you could actually even say, and we've written papers arguing this, that it's almost like upstream of mtorc1, because the pi3 kinase pathway is one of the inputs into mtorc1. In many ways, mtorc2 is less important than mtorc1. I mean, you can modulate it more and more, still survive more. So we've really focused largely on MTORC1. And when I first got here, you sort of asked me, okay, well, what did you end up doing? Right? And I was pretty worked up when I got here, and I had to realize I was sort of running a lab and unclear exactly what I was going to do. And I ended up working on MTORC1 or MTORC, I should say, largely because I didn't know anything else. So I basically had to work on something. And I remember some people here were pretty critical of me working on rapamycin. They were like, why are you working that silly molecule? Okay, now you have the target. And the truth was, that's what I knew how to do.

Even at the time, you didn't appreciate what you do now, which is that effectively MTOR sits at the center of the universe for at least some of the things that we care a lot about, including potentially longevity.

We did not.

When did that become clear to you? Is this.

That became clear you know, we tried. When we started understanding the connection to nutrients and the fact that caloric restriction had been connected to longevity, we started thinking, okay, we actually tried doing experiments on worms at the time with rapamycin. And turns out rapamyce doesn't get into worms. But there was really some. There was an important paper in worms where there was a mutant in the C. Elegans version of MTOR that had longevity effects. I would say that was sort of the key paper.

And this is unrelated to Daf2.

Unrelated to Daf2. Although interestingly, in the screens that gave the Daf mutants. One of the Daf mutants, in retrospect, one of the ones actually had never been identified what the gene was. It was simply a mutant that had a mutation. Turns out to be Raptor. I think it's DAF 15. I don't quite remember. So there was a DAF 16.

I'm trying.

I don't remember. We could look it up. Yeah, but. So it was interesting. There was all these DAF mutants that had these interesting phenotypes. And once we found raptor, someone went back and found that One of the DAF mutants was actually raptor. So that connected again to MTORC1. Now, not only were there mutations in MTOR itself, the C. Elegans mtor, but also in the C. Elegans Raptor that connected it to it. We did not realize, of course, our paper was published in Cell. Stuart Schreiber's paper was published in Nature. I remember Nature wrote in News and View. So people appreciated that the finding of MTOR mattered. But I think more from, okay, this is a new signaling pathway. This is a new component. I don't think we realize that it really. We certainly didn't. At the center of so many important processes as we do now, people sometimes joke and say, well, you know, MTOR does everything right. So if something does everything at some point, okay, how interesting is it? Right? So it's a funny.

Not a lot of people studying oxygen these days.

Exactly. Or like from the ribosome. We all appreciate the ribosome makes proteins and so it's important for everything. But you don't study it as a sort of something that's regulatable. Although now we realize ribosome is sodium transport channel. Exactly. So it starts to fall into that category. But luckily we have enough of these sort of regulatory systems that Chloe shows us it's a very regulated process in the cell.

But today, mtor and by extension rapamycin and its analogs are really interesting not just in your world, but in mine. So the plebes over here out in the peanut gallery, this is super interesting, right? This is potentially a molecule that could make people live longer, at least if what it does in yeast, flies, worms and mammals is any indication. So why is it that rapamycin, or asked another way, why is it that the inhibition of MTOR, or specifically MTOR complex 1, as you'll probably elaborate on, can extend life?

I find that a very interesting question and it's a question that I'm often asked and I think we should say up front, we don't know the answer to that question. One way of addressing it is that you can eliminate many of the things that mtorc1 does and then ask, well, now when I inhibit mtorc1, do I still get lifespan effects? If you do that and look at many different processes, probably you'd vote. Autophagy is the most important thing that it regulates. Which as you know, autophagy is this self eating process where the cell breaks down some of its own components and presumably has to remake them. And so in a kind of naive way, you might imagine that what you're doing is throwing out the old and making new. And again, naively you might think, well, that's going to rejuvenate a cell. Although none of that is of course proven. So that would be a simple answer, but it clearly is not the whole answer. So my answer to your question, why MTOR modulation has these longevity effects and yet many other pathways that in some ways are as complicated and as important for a variety of things don't. And this is the way I think about it. I think about it like I try to analogize it a little bit to like a building, right? So if I wanted to take a building like this one and make it younger, rejuvenate it, you know, I can't just get a plumber or electrician or a painter, right? Or wood, a carpenter, because the building has many different features of which all of them have aged. What you really need is a general contractor, right, who's going to then bring.

In all of those, all the subcontractors.

And fix all the subsystems. We look at an old building, an old building has lots of things that are messed up from it, from the electrical systems to the windows, to everything. And to some extent MTOR is like the general contractor for the cell. I don't know of any other pathway that does as many things, right? MTOR basically has a finger in every major process in the cell. And so I Think. Another way of thinking about your question is what's the simplest way to manipulate a cell so that lots of things are changed? And the answer to that is a modulated mtor. Because all these other pathways will, you know, maybe some of them will regulate transcription, maybe some will do translation, some are going to change the shape of the cell. But if you got to do all those things plus more, the only way of doing it with like a single hit is to go after mtor. It is like the thing, it's like the brain of the cell which then has all these subroutines that do all these things. And so to me that's the simple answer, is that to impact the state of a cell, to rejuvenate it, to slow the aging process, you can't do one thing, you can't do two things, you can't do three things, you can't do 10 things, you probably have to do 100 things. And the only way you can do all of those things with one button is to go after mtor.

Now in biology that tends to be a two edged sword, right? Because presumably if you have one switch that controls so much, if you have the wrong general contractor, or if the general contractor does the wrong thing, the effect is much more noticeable. So when did it become apparent to you or how is it apparent to you that this isn't just a linear relationship between signal and response?

This is a very good point, right? So you could say, well as a general contractor there's a lot of things. And so not only is anti aging one of the things it does, but how you sort of sperm production, which is a potential target of heart function, right? All these things require it. And so okay, you might get the anti aging effects, but you're also going to get all the downsides. And I think that is certainly true. And that's the major issue with targeting mtor.

Because at the time you really kicked your efforts off here, people thought of rapamycin and MTOR as a one trick pony, which was you give this drug every day. Your immune system, specifically your cellular immune system doesn't work as well. And at least for that subset of patients who had foreign organs in their body, that's a reasonable thing to have.

And incidentally, there is now, so funny, rapamycin started as an immunosuppressant. The interest in MTOR in the immune system pretty much was non existent. And now there's an entire field of so called immunometabolism of which MTMTOR is probably 50% of that whole field. And so it's MTORC1, MTORC2 in different immune cells. T regs, T helpers.

How much of this came out of the Novartis work from three years ago? Did this precede that or.

Well, it preceded that. I mean, the Novartis work was the first sort of work in humans. Right. That clearly showed beneficial modulation of the immune system. But in terms of studying which immune cells are most affected by rapamycin and what the role of mtorok1, that's come out of the academic world by a number of groups that were heavily enabled by the discovery of Raptor and Richter. Because now you could genetically inhibit each of those. And one of the things that my lab, we've really tried to do is to put our mice out there. And so people use, for example, our Raptor mouse or Flox so called Phlox Raptor mouse a lot. But this question of, in a way, what you're saying is how much can we sort of tolerate of mtor medulation for beneficial effects vs non beneficial ones? And again, I don't think we have the answer to that to some extent. Rapamycin is not a complete MTORC1 inhibition. We know that. And complete MTORC1 inhibition is probably not tolerated. And so rapamycin might be as good as you can get. You get some modulation.

Let's say a little bit more about that. So you're saying if we could wave a magic wand. Bobby very eloquently spoke about why inhibition of MTORC1 leads to inhibition of MTORC2 and what the temporal relationship of that might be. But I don't think we got into this issue, which is if I could wave a magic wand and completely inhibit MTOR Complex 1, not lay a hand on Complex 2, why wouldn't that be a good thing?

Because MTORC1 is probably required for the growth of any normal cell. So to make. For a cell to basically make its organelles, to make its proteins to divide, mtorok1 is probably an essential.

So at that level it would start to mimic a crude chemotherapeutic agent that modulates. It becomes 5 fu at a ridiculous dose or something that's going to basically slough off epithelial cells.

Exactly. Air falls out, cause basically atrophy of everything anti growth and probably cell death. And in fact, in many tissues, when you delete Raptor, it can be quite bad.

That's the phenotype.

Yeah, like an epithelium in the gut. At least when we've Looked, that's what's in it. So I don't think there's two issues going on here. As Bobby surely told you, rapamycin will also, with a longer time point, inhibit MTOR 2 and that is potentially bad for glucose homeostasis. The other issue is that rapamycin doesn't fully inhibit mtorok1. So in an ideal world, you'd like to have, and what I mean by that is that MTRK1 probably has dozens of substrates and arapamycin only effectively inhibits some of them and not others. Including, for example, autophagy is relatively weakly modulated by rapamycin.

Why is that?

Because the substrate, what rapamycin basically does is sort of occlude the substrate binding channel in NTRC1 and it's basically physically occluding. And depending, probably we don't, you know, this is somewhat hand waving, but there's some evidence for this. Probably the size of the substrate, if it's smaller, it might get easier and it's not occluded, if it's bigger, it's going to get blocked. And so probably the key substrates in the autophagy pathway simply are not as effective because they get into the kinase domain of mtrp1.

Still, by the way, is this issue different for any of the rapalogs? No, they're all basically producing the same effect as rapamycin.

Some people might argue differently from that, but in my experience of them, they are basically like rapamycin with maybe different PK PD properties. But from a mechanistic point of view, I wouldn't expect differences and I haven't seen those differences. But so in an ideal world, you might want a molecule that would inhibit all the substrates of MToRok1, not touch.

MToRc2, but not do it constitutively, not.

Do it constitutively, but also maybe not to 100% inhibition. Right. So I'm not sure I would use that molecule to wipe out mtorok1. I would use it to bring down all the mtorc1 activity of all towards our subject to some extent, leaving MTORC2 intact. I think that's going to be very hard to do by targeting mtorc1 itself, because mtorc1, mtorc2 share the same kinase domain. And so you can't go for the ATP binding site, which is most kinase inhibitors, mtor as a kinase, protein kinase, like leavec, for example, they all go for the ATP binding site. So we're probably not gonna do it for that. And so Our view is that the way to accomplish that is not to go after MTORC1 itself, but to go after its upstream regulators. And the big benefit in my view of doing that is that you should be able to have something now that modulates all mtorC1 substrates. And you can also start to get tissue specificity because these regulators vary in importance across tissues. The aspect of this pathway that's kept our attention for two decades at this point is that NTRC1 is basically regulated by everything. Anything I do to the cell, whether I change nutrients, oxygens, ph, growth factors.

Osmotic, what's the direct effect of glucose and or insulin on MTORC1. It obviously plays an enormous role on Complex 2.

It seems to activate them. Right. So through independent pathways, there seems to be a pathway through which insulin acts, and there seems to be a pathway through which glucose acts. And even the glucose pathway probably has several sub branches to it.

I see.

Which again, teleologically makes sense because if it's a nutrient sensor, it should be activated by nutrients. But it becomes very complicated now because you have the same nutrient acting in completely different areas with great redundancy.

Because you're looking at, in the cells that we use in culture, we can get both of these sensing systems where probably in vivo, there's tissues that are going to care more about the insulin arm, there's tissues going to care much more about the glucose arm, and there's some that are going to care about both. So if you think about being a peripheral tissue, let's say you're a cell somewhere in your leg and you need to make a muscle cell, let's say a muscle cell, you need to decide whether you're in an anabolic state or a catabolic one. So clearly there's things of use and all that, but let's say just in response to nutrition, you kind of want two pieces of information, right? One, you want to know that the organism that you live in as a whole is in a fed state. You want to be a good member of the community. And that is reflected by things like insulin, which basically tells you the pancreas.

It's a global metric.

Pancreas saw glucose, we sent out insulin.

Yep.

And the other one is you actually want to know that you have the nutrient that you need. You could have like central command telling you, hey, I got glucose, but if you don't have glucose, you can't do it.

It's a local issue.

And so you really want like the central signal and you want the local signal. So I think one can interpret that the pathway senses both the nutrient.

So the amino acid can be a local. The glucose molecule itself itself is a.

Local one for sure. We know it is.

Whereas the larger peptide can be sort of the central command.

And now you can extrapolate that to. There are many signals that are secreted in response to food, right. Insulin just being one of them. And then there are many local nutrients. And now you can start to see the enormous complexity of the problem. Right. And now you add a temporal component to it. And now you actually have tissue.

Yeah.

And then you make things tissue specific. So our view has been if we can find the sensors of the nutrients, and that's what we focused on. So we focus a lot on amino acids, but we're also working on glucose. If we could find those sensors, by definition they'll have small molecule binding pockets. Right. Because they bind nutrients which are small molecules, although they're small, Small, small molecules compared to drugs. We should build a drug.

So in 2015, in the fall, you had these two papers that came out that looked at leucine, of course, huge interest, but also arginine. Leucine and arginine can get into a cell very easily. Do they passively diffuse in?

There's transporters for them, relatively straightforward transport, high capacity transport.

Okay.

In the cytosol, these amino acids bind to receptors that then downstream result in the activation of Tor Complex 1, specifically MTOR Complex 1. People have long talked about how branched chain amino acids are important for building muscle specifically to be consumed in a workout. Was always sort of the rhetoric, presumably because that's a very catabolic time for muscle. It now seems that that makes sense, at least in the presence of what leucine's doing. Do we think that the other two branched chain amino acids are having any effect in our.

At least when we look at the receptor we found for leucine, and then we look at the concentrations at which it might bind the other branched chain amino acids, we don't think those affinities are relevant. Particularly valine is way too low. Isoleucine, maybe in some situations could act through the receptor, but unlikely. So in our hands, again, looking in a very molecular point of view, it really seems like leucine is the key one. And I would think from talking to bodybuilders and looking at bodybuilding products out there, it does seem like leucine is the one that people have focused on more than individual ones.

And tell me the difference between leucine and arginine, then, with respect to the.

Signaling is what one way of conceptualizing Amtrak one is it wants to drive anabolism. And what its goal is to detect when something's missing, for that. So we tend to think of the pathway like when we turn it on. But probably it's really key function is to turn off when something is missing. Right? Let's say you're building a house, all of a sudden you run out of concrete, you want to turn off. All of a sudden you run out of wood, you want to turn off.

And so this pathway, so the default is on.

The default when everything is there is on, but it's built, it's organized in such a way that the removal of.

Anything efficiently turns off.

Now, this is going to vary, obviously, between different tissues. And so the pathway evolved that it needs to detect leucine and it needs to detect orginine, at least in most tissues. Now, why is that? They're both amino acids. If you think about this, during the course of evolution, you're an animal that ate another animal, so you ate its muscle, you got protein. Why do you need to sense two different amino acids? And they're very structurally different, right? They're about as structurally different as you could get in terms of amino acids. We don't have an answer to that. Why did evolution do that, pick these two amino acids?

I mean, it's a phenomenal question. I don't know enough about amino acids to know what the evolution of amino acids looks like. I mean, a billion years ago, I assume we didn't have the same amino acids.

No, I think we did.

Yeah, we did.

So most all forms of life have problems.

So basically, from the beginning of when we had DNA to RNA to protein, we had the exact same amino acids. So then it's even more of a mystery. Why in the. Why did we.

Part of people in the lab that I'm sort of encouraging to look at other organisms, because the sensing part of this system is probably evolving quite quickly because different organisms live in different environments. And so, for example, flies, we know already don't care about arginine. They care about leucine, and it turns out they care about a whole bunch of other amino acids that we don't care about.

What about yeast?

So yeast, in many ways is the most mysterious, because yeast. So we don't know any sensors in yeast, and none of the sensors we have found are in yeast. And that's because yeast can make amino acids to some extent. Yeast is very primitive. You give it nitrogen, you give it carbon, it's going to make every amino acid. So things like leucine, which are essential to us, are not essential to yeast. They can make it.

In what state do yeast cease to activate tor? Only in the absence of the essential elements.

So regulation of TOR is not as well studied in yeast because it's harder to detect the output. And so typically what people do is they change the nitrogen source or they change the carbon source. And so my view is the yeast has to have a sensor of nitrogen, whatever that means. Right. It's not so easy to understand what that means. And a sensor of carbon, but not a sensor of individual amino acids. And as we find more sensors. So we now have. We've now connected the pathway to methionine sensing.

Yes.

And we have a receptor for that that yeast doesn't have that either. And so I've actually also tried to encourage people in the lab to look for what might be a nitrogen center in yeast, for example, ammonia, which is a simple form of nitrogen. Maybe that's what's sensed, maybe acetate is what's sensed for carbon. But we don't know.

So say more about methionine, because in the protein restriction literature, certainly one argument is that methionine restriction specifically could be beneficial if one believes that low IGF is beneficial. And we can talk about whether that's causally the case or not. Not even getting into the IGF binding proteins. Where does methionine fit into tor?

Right. So methionine actually is a very interesting one, as you said, there's extensive literature on what's so called methionine restriction having quite beneficial effects from glucose homeostasis, actually to quite very reasonable lifespan extension effect as good as caloric restriction. And there are some papers in flies, genetic papers, that suggest that some of the methionine restriction effects go through the TORT pathway in flies. We got to this basically through the protein. We found a protein of unknown function and we tried to figure out what it did, and it turned out to be a sensor of this metabolite called SAM S Adenosylmethionine, which is basically made by methionine. So it's actually quite interesting.

And people supplement with a variant of sam.

SAM exactly right. SAM actually has some pretty interesting clinical effects. I see some quite convincing data on antidepressant effects of SAM out there. So this is the sensor here is interesting because the other sensors we have directly bind leucine, directly bind arginine. This one doesn't bind directly to methionine. It binds to a Metabolite made by methionine, which is sam, which sam. Many things can feed into sam, so it actually can integrate lots of signals. So this sensor basically behaves like the other ones. As soon as methionine levels go down, SAM levels go down. This sensor therefore inhibits this pathway.

So SAM would not be a longevity agent by the oversimplification that excess SAM would be akin to excess methionine would be akin to failing to inhibit tor.

Exactly. So methionine restriction could be presumably rescued by giving sam. And we actually know in the pathway that we've built in cells that that's true. You can bypass methionine simply by giving sam. So a molecule that could basically trick this sensor into thinking that SAM was not there would be quite an interesting one. I think methionine is probably the most interesting of these amino acids because if you fast an animal, methionine is the amino acid that drops the most. And the reason for this.

And you looked at all of the amino acids in mice.

Okay, so we should do some of this in humans, but it kind of makes sense.

I can volunteer if you want.

Sure. Well, we could definitely profile.

Yeah.

The reason probably is that arginine, you can make some. Right. Your liver can make it. And then leucine is an amino acid that's an essential amino acid, but to some extent it's only used to make protein. That's it. So when you fast, you start to break down your muscle and release leucine. Methionine is not only an essential amino acid that you use to make protein. And remember, the first amino acid of all proteins is the methionine. So by definition, every single protein has methionine, but it's also incredibly metabolically active through SAM and the so called methionine cycle. So when you fast, you probably just can't generate enough methionine by breaking down your proteins to keep up with methionine demand while you can for leucine. So if you look at the blood of an animal that's fasting, methionine is the number one dropped amino acid.

Do we think that's true in autophagy in general?

What do you mean in autophagy?

If we put an animal into a state that induces autophagy independent of caloric restriction. So for example. Yeah, yeah. Would we see the drop in methionine as a readout?

You know, you might expect it to go up actually. Right. Because autophagy is going to break down protein. You Might methionine?

Yeah.

If you're not recycling.

If you're not recycling. And it depends if you induce in the state, for example, post exercise. I don't know what we know about the use of methionine in sam. Right. Are you doing a lot? So SAM is used for methylation reactions, and there are hundreds of methylation reactions. SAM is the second most common CO factor in enzymes after ATP. Everyone knows about ATP and ATP is energy, and then it's used in many, many, many reactions for phosphorylation, but SAM is the second most common one. So there are literally hundreds of proteins that use sam. So maybe after exercise a lot of SAM is used. I don't know. It's an interesting question. Right. But with fasting, methionine definitely plummets. SAM definitely plummets. And so we're now generating the right animal models to ask whether the sensor we have is involved in the effects of methionine restriction. So we can basically knock it out and then do methionine restriction. And if the animal doesn't have the health benefits of methionine restriction, it means that this sensor and by extension MTORC1 are the key mediators of a methiro restriction. So we'll see.

So, coming back to rapamycin specifically and all of its limitations, so we've established that you can't just take rapamycin all day, every day, because that experiment's been done. That's the clinical utilization of it. Certainly the animal data have suggested, and the human data have suggested that an intermittent dosing of rapamycin could produce a beneficial phenotype with respect to longevity specifically and also with respect to immune function. So if you had to guess based on triangulating these data, assuming no new drug came along that was going to selectively do some of the things that we've discussed, how would one dose in an animal, or a human for that matter, rapamycin to increase the odds in favor of longevity and against harmful side effects, which presumably the most obvious ones would be immune suppression and. Or glucose homeostasis disruption.

Yeah. And also epithelial sort of toxicity, particularly the GI epithelium. So I think the intermittent approach is definitely the one that makes sense, because if you buy the idea that you want to induce autophagy, which a lot of people, of course, like yourself, who study the effects of fasting, also view that that's one of the goals of fasting, is to induce autophagy. So if we basically want to chemically induce autophagy without fasting. I think the intermittent dose is what makes sense. You basically let have an induction autophagy, a relatively weak one with rapamycin, but then let the system rebuild. It's clear that both mtor you need just right amounts. Right? You can't have too little, it's toxic. You have too much, it's toxic. The same thing with autophagy. If you remove autophagy, it's really toxic. If you have too much autophagy, it's really toxic.

Cycling, anabolism, catabolism might be the single most important thing to do.

It might be right. And I think it's, it's hard for us to know, but those intermittent dosing strategies, the every other day feeding strategies all point to that. And the genetics where too much is bad and too little is bad, also point to that. Right. So if you genetically inhibit this pathway by deleting raptor, if you genetically activate it by deleting these repressors called the tuberous sclerosis complex, both are bad. Both in fact in many tissues, like the muscle give the same output, they get muscular dystrophy.

Yeah, I was just about to say there's an overlap with muscular dystrophy here, isn't there?

Exactly.

So this may be a theoretical question, but when we think about the life extending properties of rapamycin, do we believe that it is a result of delaying the clinical onset of disease? Let's use a disease where that tends to be more binary, like cancer. Obviously, cancer spends probably 70 to 80% of its time undetectable. But due to just the law of growth, it becomes detectable only at the end. So do we think that in as much as say, taking these agents would allow you to live longer by not dying from cancer at the same period of time, does it delay the time it takes for cancer to become clinically detectable and or delay the demise of the animal once it has that cancer?

Yeah, I think specifically in the case of cancer, rapamycin is. There are some situations where it has some decent activity, but in general it's not a cytotoxic agent. Right. It's not going to kill a cancer cell. It's really going to.

Once an organism has cancer, do you know if it's doing anything to prevent the development of cancer?

We don't know that. Well, and the only. There actually has been some epidemiological data where people have compared cancer rates in.

Transplant patients, identical patients who are with.

And without rapamycin 506 versus rapamycin. And it's actually quite interesting because. Because, as you know, immunosuppression in general is associated with higher cancer rates. Right. The idea that you have less immune surveillance, that's not seen with rapamycin. So it is seen with FK506, it's not seen with rapamycin. And the argument has been that rapamycin itself has cancer cell autonomous, independent of.

The immune modulation problem.

So you're presumably getting less immune surveillance because it's an immunosuppressant. Although of course, that's not proven. But you're mitigating that by now directly.

And they've canceled each other out.

They cancel cancer.

And you know the size of the effect from the FK506 cohort.

Exactly. And other immunosuppressants things. Cyclosporine, have also been looked at that. So my bet would be that in the case of cancer, you're not gonna. You're not gonna.

You're not gonna cure cancer once you've got it.

But you.

But I don't think you're gonna modulate the incidence like the mutational frequencies that are giving you cancer. Right. So if you think of cancer in a way is easier to think about when it starts because you say, well, it starts when you have a cell that has all the requisite mutations to be.

To evade detection.

Exactly. It has uncontrolled growth. So if that's the point it starts, I think we're not going to affect that. But once that cell exists and now has to start growing and also escaping the immune system, I do think that's probably what you're going to affect in other diseases, like cardiovascular disease, where you could imagine things like autophagy could be quite modulatory. I think you can imagine that you're also affecting the incidence. The exact point at which you'd say, okay, this is an atherosclerotic plaque or not.

What do we know about rapamycin and TOR in the brain, especially with respect to neurodegeneration?

Yeah, that's a really interesting one. And that probably is a really important question for the future. So we know autophagy matters a lot in the brain if you delete autophagy. And really Mitsushima was the person who kind of made autophagy interesting to lots.

Of people and it was awarded the Nobel Prize.

No, no, he wasn't. Mitsumi was for original.

He didn't share.

He didn't know. Which I think was a bit of an oversight in my view. But anyhow, he basically studied autophagy in the brain made mutations, showed you got neurodegeneration. Right. So that was a really important finding. Connects up to lysosomal storage diseases, which autophagy, basically the autophagosome fuses with the lysosome. So now you have that connection. So I think like in all tissues, it's a bit of a double edged sword. You clearly need MTRC1 activity to maintain healthy synapses. Certainly during brain growth. You do. If you make mutations ramble in a growing animal, you basically don't have a cortex. Right. On the other hand, you clearly need to be able to modulate MTORC1 to have some level of autophagy to keep the system healthy. Now you could debate is that in neurons, is that in glia? It's probably in both. People have made mutants in certainly in neurons, which suggests it's both. But then some of those promoters are a little bit dirty. But the real question in the brain is what modulates MTORC1?

Because it's not probably nutrients because they're so constant. You mean like exactly.

Your brain, Your body.

Yeah. Your brain prioritizes nutrients in the brain over it.

It basically protects your body. So if you take an animal and you fast it for two days, a mouse, it loses a lot of weight, 25% of its weight. And now you take every single tissue and you weigh it. Every tissue has shrunk except the brain. Some, like the thymus, have shrunk, ridiculously. The kidney shrinks, which you wouldn't expect. The heart shrinks, the brain, nothing. Now clearly, probably if you, in a mouse, you can't do that extreme of fast. And so the body protects the brain from a nutrient point of view. Yet MTRK1 activity is high there. Clearly we know that we have to modulate autophagy, so something must be inhibiting AMTROK1.

By the way, this is my peripheral argument for why, and I'm in the huge minority here. I do not think the brain is really the appetitive center. I think it's the modulator. But I for that exact reason think it wouldn't make sense for evolution to put our appetite center in our brain. It should be in the periphery, it should be in the liver. I think, I think the liver should be in the.

People would argue that things like the hypothalamus are in the periphery. Right. Because they're not protected. There are parts of your brain like the hypothalamus.

Yeah. The point is, I think it has to be. Your appetite center needs to be regulated to something that's senses very rapid change.

The outside, for sure.

Yeah, for sure.

And exactly where it is and you know, and the bottom line is probably.

But I never thought of it through the lens that you just explained it, which was. The implication of that for TOR is enormous. So does TOR look different in the brain or. I mean, obviously the protein won't, but do the CO factors around it look different?

You know, we keep talking. We have never done framble biochemistry out of the brain. It's something that would be very interesting to go and do now. I think now it's something we talk quite a bit as a lab to do. We haven't quite done it at all. But then what actually regulates it? It's very clear that neuronal activity does. But are there, like as you're suggesting, maybe neuronal specific factors regulate. I think that's a completely open area. I've tried to get some of my students interested in that. My brother's a neuroscientist. He's argued we should really do some work there. We just haven't. Maybe when we run out of sensors in the periphery, we'll go to the brain. And that's where I purified mtor. It was out of the brain. So there's a ton of MTOR in the brain. And I did that not because I was like whatever. I basically measured how much there was and it was clear the brain had the most.

One of the challenges of studying biology in humans is that you can't do the same experiments you can do in animals. If we had a gedankin experiment where you could take a sufficiently large number of human subjects and divide them into groups and you. So you had a control group. These guys were gonna do everything that the standard American does. You had a group that you could give rapamycin to in any way, shape or form. You decide. And then you had a group in which you could manipulate their behaviors and they would behave as animals. They would do anything you want with respect to how they would eat or how much or when.

Exercise, whatever you like.

First question is, how would you design arms two and three to have the best outcome with respect to longevity? And then I'm very curious to know what you think the difference between groups two and three would look like.

So I think as we spoke before, I mean, that MTOR modulator arm would probably be an intermittent type dosing one where hopefully we'd have biomarkers of that. And you and I have spoken in the past. A biomarker for autophagy. For example, a biomarker for MTOR activity, and you have to decide what tissues you cared about. Probably the muscle would be one that you'd want to focus quite a bit on, and perhaps the liver. Now, I don't think that MTOR modulation on its own is going to give you all the benefits of good lifestyle modulation. So it might give you lots of the benefits of the dietary manipulations, the fasting manipulation, although clearly there's differences there. But I'm not sure if AMPOR will give you all the benefits of the exercise modulation. Right. And so if on the lifestyle side, which you obviously know better than almost anyone, what you'd exactly want to do, there'd clearly be an exercise component to it on top of a dietary component. I think MTOR modulation will give you a subset of that.

I see.

And so.

So let's simplify the experiment then. Let's assume that everything but food is the same in the groups, and the RAPA group gets the intermittent dose as you see fit. And the other group now can fast or do any sort of CR mimicry that you want. Do you think that normalizes the playing field?

I think it gets a lot closer for a simple reason. So if you give an emtromodulator versus a fast, remember there's one really important difference is that nutrients in the MTOR modulation case are actually still high because the person's not fat. In fact, if you actually look in cells, they can actually even be higher because the cell thinks it's starving. So it does all this stuff, it.

Shuts down processes that would accumulate and.

It up regulates more accumulation. And so we've looked in cells, so actually they tend to go up versus a fast, where things are going to be lower. On the other hand, if all those nutrients are eventually doing their stuff by communicating through MTOR and you've sort of inhibited downstream to those downstream processes, things look the same, doesn't matter. We have a lot of nutrients here and very low nutrients here. So the modulation of ambulatory is what matters. And so I think to answer that question, we really need to understand whether all these nutrients, which are still there in the fed state, have a lot of other signaling effects. And it would be naive to think that they don't. They do, we know they do. Now, do they matter? And do they matter a lot? I don't think we know. And I think a lot of the genetics and pharmacology would argue that within the range that we can actually manipulate lifespan, it could be that those fasting regimens and Rapamycin are somewhat similar.

And certainly in the mice, it appears to be at least similar, if not better in favor of rapamycin.

Exactly. And that's why I'm particularly excited about the methionine restriction work. Because caloric restriction is not only hard to do in people, it's hard to do in animals too. It's really hard. You have to weigh the food pairwise. Feeding, it's a real pain and it's a real restriction to doing lots of experiments. While methionine restriction is a lot easier.

Yeah, you just buy methionine free chow.

Yeah. Not totally free, but lower. Right. And so there we can do these kind of experiments where you could do methionine restriction plus rapamycin. Right. And actually ask, do you get synergy? Do you not? So, so I think that's going to be an intervention that's going to be a lot easier for us to play with.

So if resources weren't constrained, what are the sort of dream experiments or what's a dream experiment that has been on your mind that you want to do, but it's either technically we're not there yet, or it's just economically a challenge.

Yeah. I think what I want to know, and this is, I think the challenge for anyone who does what we call signal transduction in a dish, like we've done for a long time, is to really try to understand in each different tissue in a temporal fashion, in response to a variety of different diets and nutritional states, what those tissues are actually doing. Right now we have these little time points in the liver, in the muscle, we don't really have a deep sort of kinetic understanding of what the actual physiology is doing. Right. We'd really like to know, because even.

In the mouse, you know, phosphorylation, in one moment you don't have an integral tissue.

We don't have that. And we don't even have. It's just they're expensive and hard experiments that do. Let's say I was really wanted to take mice, fast them and in all different tissues. And ideally, you know, tissues are complex. Right now with all the single cell sequencing, we're seeing much more complexity. So even in tissues like the liver that we tend to take a chunk and sort of say it's liver, we know that amazing complexity. Right. And so in ideal world, we'd like to have a description of what all these different tissues are doing over time. And then you'd like to do it under different diets, under whether they were obese mice, whether they were exercising mice. And so the matrix becomes ridiculous at that point. But I think that's the future of signal transduction. People like me have done a good job of finding all the pieces in some random cell line in a dish. And clearly the systems have all these pieces because allows them to communicate in vivo to many, many different upstream signals. And now the challenge is how do we go back and actually see that happening? And that's going to teach us, okay, which tissues actually matter. We've talked a lot about longevity. Do you need to impact all tissues? Is it the muscle, is it the liver, Is it the brain? Maybe that you need to impact people debate still how much rapamycin gets in the brain. Are you actually affecting the brain? I don't think those are open questions to some extent. So it would be a complete description of what these systems are doing over time across many tissues under many different states.

Well, David, we're pretty much out of time, but is there anything else that we should at least take advantage of while you're here?

I think we already touched upon it when we Talked about targeting MTORC1 or other things. And so I think to me, and this is why we sort of had commercial interest in this regard, how do we go and target other things upstream that might be more amenable to giving us sort of more this dream molecule of a pan MTORC1 inhibitor and no MTORC2 activity.

David, thank you very much.

Thank you very much. It was a pleasure. Thank you.

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