Rhonda: Yeah. And I think that was the big eye-opener for me when I read this study, I don't know, a couple days ago. And it wasn't a new study but, you know, it was like, "Oh, well, this changed really dramatically." But then it wasn't, like, a permanent thing, I mean, it went back. And so, yeah, it's almost like you're saying with interventions, it's like, "Well, I mean, make sure you didn't get sick," or, like, you know, sick, like, too early before, you know, measuring. And we can talk about that in a little bit, a little bit later, but to get sort of just back into the cause and effect of aging and if the epigenetic clock changes are really causal, I mean, of course, you're, obviously, trying to figure that out, but, like, even if it was, let's say, downstream of something, if it was biomarking aging, what...the epigenetic changes that are happening with aging, you kind of mentioned this early in the podcast about how, you know, they're clustering in gene regulatory regions, and, so, they're changing the way genes are turned on or turned off, is there like a feed-forward loop in aging where it's like, "Okay, now these epigenetic changes are turning off genes that we want on to repair damage and they're turning on genes that are cellular senescence?" you know, so, it's accelerating this feed forward loop...

Dr. Levine: Yeah. I mean it's definitely possible. I think, yeah, it's really hard to figure out causality here, right? Like, and it could be that, you know...I mean my perspective is not, "There's a cause of aging," right, and, you know, "it's this thing and, once you fix that, everything else will go away." I mean so many things go wrong and your system can change, it can diverge in some...going back to kind of Mike Snyder saying, "Even if you bring that down to the molecular level, there's so many different ways that someone's system can kind of change over time." And I don't think it's just this one thing that's going to then drive all of aging.

And yeah, you know, our systems are responsive, right, so, one thing changes, something else is going to respond. And that can be maladaptive, which would, you know, snowball things. So yeah, I think it's going to be hard to figure out like what's causal, what's correlative, but I would say, even if it's not what some people might consider the central driver, as long as it's picking up things that are critical to aging and you can use that to track aging or understand it a little bit better, I think it still has utility. I don't know if it needs to be kind of the central cause of aging for it to be useful.

Rhonda: Right, exactly. If you can track it and/or use it for basic science, you know, to understand the processes better...but with some of these genes, I'm just curious, do they know, has research shown, your research and others' shown that, as the epigenetic clock ages, we are seeing more genes that are regulating NF-kappaB turn on causing more inflammation? So, it's not necessarily the cause of aging but it's helping accelerate it as you start to accumulate these epigenetic changes, as they start to shift.

Dr. Levine: Yeah. So, the hard thing has actually been looking at the genes that these CpGs are assigned to or that they collocate with. And actually that's been a little bit less clear because the methylation patterns are not, as we might expect, correlating with the expression patterns. And there's a number of reasons that could be, it's because we're looking at lots of cells in the population, you need to look within an individual cell to actually be able to see this. It could be, you know, there are other epigenetic modifiers that could also be important in this, so, it's not a one-to-one. But we can just look at general gene expression that epigenetic clocks are associated with. And, you know, you can kind of find certain pathways that seem to be important. And some of these are inflammatory, or other aging pathways that we'd kind of expect.

But yeah, we still don't know exactly what the CpGs that are in these clocks are functionally doing. Even though we say, "Oh..." I actually said it in the beginning of this talk, when you have methylation, it's repressive, when you don't, it's active. But it seems like it's actually a lot more complicated. And one thing that I always come back to is I can make a clock out of a few hundred CpGs that are, you know, they're in specific genes, I can remove all of those genes and remake a new clock and I can get the same clock from a totally different set of genes. I don't know what that means in terms of understanding the functionality of what we're trying to capture but I think it just suggests it's not as simple as, you know, these 20 genes are turned on and these 20 genes are turned off.

Rhonda: Right, right, a lot more to learn. Epigenetic age reversal, that's a big interest, of course. And I'm sort of curious about, like, some of your thoughts on some of the...there's been some recent studies. So, we were talking about programming, right, we were talking about, in a way right, with the developmental program and the epigenetic clock sort of really tracking that well, being part of that, in some way connected. I don't exactly understand why [inaudible 00:42:11]. But I don't think anyone knows. Okay. So, some of this work with interrupted cellular reprogramming, or the partial reprogramming, as it's called, a lot of the works come from Juan Carlos Belmonte's Group where they can...maybe you can explain like what this is to people and how that affects epigenetic aging or what's known or not known?

Dr. Levine: Yeah. So, this really came out of work, originally, from Shinya Yamanaka who discovered, what we call, these Yamanaka factors, which are four transcription factors, we just call them OSKM, which, when expressed, you can actually take a somatic, so, an adult cell, and convert it back into what looks like an embryonic stem cell. So, we call these induced pluripotent stem cells. And then you can use those to make a number of different types of cells.

But the interesting thing and why aging researchers got really invested in this science is that, not only are you making it embryonic-like in terms of its stem-cell properties, but the epigenetic clocks seem to be almost completely reversed. And we've actually shown recently they're not completely reversed but you can take a skin cell that has an epigenetic age of 40 and do this, it takes, you know, a few weeks to do, and basically get back to an epigenetic age of zero in those cells.

Rhonda: And you keep it the skin cell, it doesn't lose its identity?

Dr. Levine: Yeah, so, it loses its identity...

Rhonda: Okay, when [inaudible 00:43:45] back.

Dr. Levine: Yeah, so, this is considered kind of this full epigenetic reprogramming. And then what Juan Carlos Belmonte and others have done is look at this idea of partial reprogramming. So, can we push the cell back a little bit? Because actually what we find is that this age reversal happens first prior to the cell losing its identity. So, can you do that part without pushing it all the way back, what we consider up or down the landscape, to this pluripotent stem cell? So, can I just make an old skin cell a young skin cell but still a skin cell? So, that's the goal.

Rhonda: And with some of the recent work, at least out of his lab, they're using a premature aging mouse model, a progeria model, and have shown...I know there's a new publication I haven't read, just came out, but the older one, the first one, 2016 or something, cell paper, I remember they showed, in multiple different organs, it seemed to reverse some of the hallmarks of aging, you know, and the organs were performing functionally a little bit, you know, younger than you would imagine, at least in this premature aging mouse model, and I think even health span of this mouse model that's prematurely aging, it seemed to be improved. I mean, what that means for humans, that are not mice, with premature aging syndromes is to be determined but the epigenetic clock was also reversed as well. Right?

Dr. Levine: Yeah. And I think the new publication, which is done in more of a wild type, not a progeroid mouse, does show kind of some reversal of the epigenetic clock. And you can do this just cells in a dish, we can partially reprogram them and show reversal of the epigenetic clock and other functional improvements in the cells.

Rhonda: So, to you, what does that mean, like, that you can do that?

Dr. Levine: Yeah. No, I mean, I think this is the most fascinating thing. Again, I don't know in terms of translation, like actually making this a therapeutic, and I don't even think people were at the point where [inaudible 00:45:41]. But yeah, I just think it's so amazing. I mean even the original thing, that you can take, you know, a skin cell and turn it into an embryonic stem cell. And just we always think of, you know, like, this is one direction, cells are going to only move, you know, what we consider this landscape, in terms of their states, and they can only go from this state to that state. The idea that it can go back I think is amazing, and I think just understanding how that process works...

And then the other thing we're really interested in is what are the features of this programmed cell? Like, does it truly look like a young cell or is it a totally different type of cell that, in nature, maybe we haven't even seen? And what does that mean for how it's going to function?

Rhonda: Totally. The questions I have on my mind are, "Okay, well, you take this, you know, 40-year-old skin cell," as you mentioned, "and let's say you're going to completely reprogram it to a stem cell and your epigenetic age goes back but, like, what happens to all the damaged mitochondria? Are they still there? Like, what about the pieces of DNA that, you know..." Like, is that stuff still there? Like, where does it go? How does it go away if it does?

Dr. Levine: The exciting thing is actually the mitochondria seems to also be kind of rejuvenated. I mean, I don't really like that term, rejuvenated, but it seems to be kind of set back to a better functioning state.

Rhonda: Oh, really?

Dr. Levine: Yeah, again, it's not clear how all these things are linked to each other. I think the other question though is, you know, cells also build up kind of these aggregates and other, you know, nasty kind of byproducts that accumulate. What happens to them? I don't think we know that. But it's an important thing I think to figure out.

Rhonda: So, I mean, if the epigenetic clock, because it's controlling gene expression, it's like, well, maybe the nuclear coded mitochondrial proteins, maybe everything's just bouncing back to how it was and, so, you're building better mitochondria. Right? I mean...

Dr. Levine: Yeah, I have some colleagues who argue that it starts with the mitochondria, getting rejuvenated, and then that's how everything else...but yeah, I think...

Rhonda: Yeah, it's back to that hallmarks of aging, you know, mitochondrial dysfunction...I mean or, as you mentioned, it's probably not just one thing, it's a combination of all these factors together combined where your proteins are misfolding and your mitochondria is functioning and, you know, your DNA damage is accumulating. And so, yeah, I mean it's a fascinating area. And the programming part, like, I was just curious what your thoughts are in terms of the basic science? Like, to me, it goes back to, again, that program. Like, there's something going on, we don't quite understand, but it's something.

Dr. Levine: Yeah, this comes back to this whole idea that I don't think what we see with aging is just random stochastic damage or errors. I think we've always thought of aging as just accumulation of errors but it really might just be a program that kind of goes wrong and there's nothing evolutionarily that needs to prevent it from doing that because it doesn't benefit, you know, fitness to prevent that program from going wrong. But the idea that it can be reprogrammed, again, using the operating system kind of analogy, that you can just take an, you know, operating system that's not doing well and do an update and take it back to this better state...and again, we need to figure out exactly what that means, but I think it's really exciting.