At Ending Age-Related Diseases 2021, Irina Conboy discussed the effects of plasma dilution and heterochronic plasma exchange and their relationship to epigenetic alterations.
Script
Thank you for inviting me again, it’s much appreciated. This is an excellent conference series. I will start by telling you that it is my deep belief that the path to rejuvenation is through improving tissue maintenance and repair, not trying to prevent tissue damage. We cannot really win against the second law of thermodynamics, but fortunately, that is not needed.
Here is a cartoon example that a young and old person experience a similar degree of damage. In fact, young people can experience more damage, particularly if old people are taking good care of themselves. What is different is that there is much better repair in the young individual as compared to old. All our aging research avenues are focused on this idea that we should improve repair capacity and regeneration in an old mouse or old person, then we will rejuvenate them.
This notion is summarized by my favorite cartoon that I usually present and most of my talks that demonstrate that at three years of chronological age, a rat will be biologically very old and full of disease, but a squirrel, which has a similar size, even higher metabolism, and a very similar diet, will be biologically extremely young and healthy. They experience the same degree of damage, they live in the same environment, but their biological age is very different, which we hypothesize is because they have different repair efficiency.
Important for my talk is to understand our axiom that, we look at several key organs, and we show that they become significantly younger and healthier but do not do the lifespan studies. The axiom means that when your organs are younger and healthier, you yourself are younger and healthier and will have a longer life. That is a given. The opposite is not necessarily true, because in many life extension studies, there will be sick animals, very, very sick and decrepit, and shivering in the corner of their cages, but they will be still in a positive column of living longer.
The second fact is that younger, healthier tissues are really impossible without everything good inside those tissues. If the tissue is young, then the cells are young, if cells are young, then the metabolome is young, and organelles and proteostasis and exosomes and gene expression, epigenetics. That is a well-known fact in fundamental biomedicine.
I’m going to introduce you to this parabiosis cartoon on a glass half full, half empty. This is blood heterochronicity: a half young, half old glass. It is important to visualize it because it explains what happens if you parabiose a young animal to an old animal, or an old animal to a young animal, and exchange their blood. In each situation, the same thing happens if you look just at the systemic milieu, you have two glasses which are half full with young blood, half full with old blood. Regardless of what you do, you always have half young blood, half old blood.
Interestingly, in vitro experiments, if you mix young and old blood together, it shows that old blood dominates. Old blood is completely dominant in vitro, and it will inhibit all of the positive things that young blood can do. There are other additional studies that suggest the same paradigm that really what we need to do with this half young, half old blood glass is to get rid of the old blood instead of adding young blood on top of it.
However, intuitively, many people assume and keep assuming that all they need to get is young blood and somehow adding young blood to an old individual would work as a medicine. These assumptions were initially precipitated from heterochronic parabiosis experiments which are shown here, again, as a cartoon.
This is the Halloween party at the Buck Research Institute on Aging in the bottom, and this is Eric Verdin’s slide. Eric Verdin is the director of Buck Research Institute. In the parabiosis experiments, we demonstrated that age is malleable. In a few studies, with Mike Conboy and myself in the Tom Rando laboratory, we show that for liver, muscle and brain, and then later on, many excellent laboratories took it forward and demonstrated the same with respect to neuroplasticity and cognition, spinal cord, heart, kidney, bone, cartilage, skin, pretty much every single organ.
It was very tempting to interpret simplistically, as shown in this cartoon on the right, that aging really is about the young blood and all you need to do is to identify a fraction or perhaps a factor. What you need also to realize is that in parabiosis, it’s not just the blood that is shared but also organs, environmental changes that are the result of adaptations to being joined together and running together in a cage for about four to five weeks.
In a cleaner experimental system, which is called small animal blood exchange or apheresis, There is no organ sharing and animals are not sutured; they do not share everything for months. It is well controlled, and it is, moreover, FDA approved.
In this experimental approach, where we were able to clearly distinguish the situation with the glasses of blood, what is important for rejuvenation, getting rid of old blood or raising young blood on top of it? I would like to mention that it was Aubrey de Grey and SENS Foundation who basically convinced us to use this approach. Not only that, they also provided very important pilot funding for developing this experimental system.
To summarize, as I presented at the previous Ending Age-Related Diseases conference last year, we published that all of the outcomes, rejuvenative outcomes, that are in our original heterochronic parabiosis paper are not just repeated, but are actually stronger rejuvenative outcomes if you simply dilute 50% of old blood with 50% of saline and albumin, returning blood cells back to the same animal.
The cells are still old, and 50% of the blood is still old, but 50% of the plasma is now replaced, is diluted. There was a rejuvenation of muscle with reduction of fibrosis, rejuvenation of liver with reduction of fibrosis and adiposity, and improvement in hippocampal neurogenesis to the degree that made all the animals statistically the same as young. I would like to emphasize here that the procedure that we have used is miniaturization of the FDA approved heterochronic plasma exchange.
It’s kind of interesting, right? It shows that young blood or young factors are not drivers of aging or rejuvenation. This may be a surprising conclusion, but it is the one that seems to be correct. That putative cause, young blood, doesn’t seem to be a cause for the effect, which is rejuvenation. Instead, we need to either remove or neutralize age-elevated plasma proteins.
Moving on to more recent findings, I’m going to bring your attention to additional phenotypes and outcomes that have been studied via young plasma, or young plasma fractions, or parabiosis, specifically, cognition and neuroinflammation. The second point that I’m going to discuss here is that sometimes, after we published our original paper on plasma dilution, people started to think that perhaps all you’re doing is removing SASP that is produced by senescent cells.
We decided to compare side by side, how does plasma dilution’s rejuvenative effect compare to the use of senolytics? Is it the same or not? In the first table, you see on the left, it basically performed the same as plasma dilution.
By the way, we also do young plasma dilution, which does not age young mice right away. That points towards the same conclusion that aging is not driven by the lack of young factors. We diluted 50% of them, but young mice still remain relatively young. They’re able to buffer that dilution, by, perhaps, higher expression of particular proteins and their production and secretion.
Now from that deviation, going back to the more recent study that we published in Geroscience, also last November, November 2020. If you dilute 50% of all plasma in old mice, they become much better cognitively, based on novel texture recognition and novel object recognition. Typically, old mice or old people are not really curious, and they do not care if it is a young object or old object, and they don’t have enough short memory to even remember which one is what. That is illustrated by this drastic difference between young/young or old old animals. After neutral blood exchange, old mice now are statistically the same as young in the aspect of recognizing new textures or new objects.
When you look at the facts of the senolytic ABT263 as compared to the neutral blood exchange, it became obvious that, very interestingly, not only the senolytic diminishes brain senescent cells, but neutral blood exchange has the same capacity to diminish SASP, beta-gal expression, as assayed by beta-gal-positive cells in the brains of old mice.
Neuroinflammation, which is another hallmark of aging, became dramatically decreased in old mice by a single procedure of neutral blood exchange. You can see that in the control animals: control young/young, young mouse exchanged with young blood, control old/old, old mouse exchanged with old blood, there are numerous CD68-positive, inflammatory microglial cells in their brain. In the paper, we described the regions of the brain where we found those. A single procedure of old plasma dilution reduces the numbers of the cells. Now, they are not much more than you would find in a young animal.
Compared to that, quite surprisingly, senolytics do not do that. If you look at the effects of ABT263, either visually or by quantification, and either in uninjured animals or injured animals, it doesn’t really matter or have a statistically significant difference in neuroinflammation. Specifically, that neuroinflammation remains high, and it is not diminished by senolytics.
We did, however, find that the CD68 signal on each individual microglial cell becomes attenuated by that senolytic. Even though the same numbers of cells seem to be activated microglia, how much they are activated perhaps is slightly diminished. Comparing side-by-side effects of neutral blood exchange and senolytic-only neurogenesis, another, similar kind of observation emerges. It’s that neutral blood exchange enhances neurogenesis of all the animals.
This is control, and this is a visual representation of animal hippocampal neurogenesis, so the animal that underwent neutral blood exchange. The red dots are the neural precursor cells that are divided. So, that is improved. However, if we use senolytics, we do not see any improvement, and the numbers of proliferating neural precursor cells remain low, about 200, and we see these down here.
So, what’s going on? It’s kind of interesting to compare them side by side, because quite often, intuitive conclusions in biomedical science are not the ones that are accurate. It’s important to note that neither ABT263, the senolytic, nor neutral blood exchange do anything to cross the blood-brain barrier.
We know that ABT263 ablates senescent cells, and interestingly, we see that there are lots of senescent cells in the brain when we use this senolytic. Even though the molecule itself does not cross the blood-brain barrier, the logical conclusion would be that reduction of senescent cells in the periphery would result in less cellular senescence in the brain. There are some molecules, perhaps ours or perhaps senescent cells themselves, that cross the blood-brain barrier and eliminating them on the periphery is reducing brain senescence.
However, that simple reduction of brain senescence does not translate in either improved neurogenesis or diminished neuroinflammation. It has some positive effects, but they’re not global. In contrast, dilution of the whole systemic milieu, we don’t know what happens to the senescent cells in the periphery. We are doing the studies, and it is quite possible that neutral blood exchange is senomorphic, so the cells are not killed, but they just become healthier and less senescent.
Once you dilute the systemic milieu in old animals, we hypothesize that there are a number of modules that do not cross the blood-brain barrier because they’re not elevated. Additionally, the leakiness of the blood-brain barrier itself, that is known to be increased with aging, could become less so. The blood-brain barrier is probably healthier, which I will mention in the next slide.
Importantly, what we also know is that even though neutral blood exchange aids in the periphery, it diminishes central brain senescence and can simultaneously increase neurogenesis and diminish neuroinflammation. Overall, the effects of neutral blood exchange are not the same as the effects of senolytics.
Seaking of the blood-brain barrier, this is my next-to-last slide. I would like to introduce a fresh off the press paper that we published with our favorite collaborator, Professor Kiara Aran shown here. It just came out a couple of days ago in Advanced Science and tries to apply the heterochronicity phenomenon to an organ chip and specifically a blood-brain barrier organ chip. Kiana did very brief and spectacular postdoctoral research in our laboratory and then moved it almost four years ago to lead her own laboratory at Keck Graduate Institute.
She is a very insightful and smart bioengineer and engineer who focuses on bioanalytical devices. She generated the organ on a chip blood inverter, where they’ve brought brain microvascular epithelial cells, pericytes, astrocytes, and neurons. This is actually what we plan to do right now; what we published has a little bit simpler neurovascular cell interface.
What this organ chip allows one to study is physiologic shear stress. It is a microfluidics chip. You can study the effects of slow versus fast physiologic shear stress on the blood components. It also allows to measure the transepithelial electrical resistance, or TEER, of the blood-brain barrier, which is the signature of its permissiveness or integrity, and also nanoparticle transfer. It has some other interesting digital gadgets, which are all integrated into an interesting, sophisticated device.
What is shown here and the data that we recently published is that if you look at the young blood-brain barrier, and compare the effects of sheared or non-sheared young and old blood components, in this case erythrocytes either in mice or in people, then particularly under physiologic shear, you see that particularly old red blood cells or erythrocytes perturb integrity of the blood-brain barrier, which is true if you look at the nanoparticle transfer, these fluorescent nanoparticles, or if you look at TEER. TEER diminishes dramatically.
This is the effect of old blood components, in this case red blood cells, and when they are experiencing physiological shear. Importantly, it is not true just for the mouse, but also for the human. In general, this BBB organ chip allows to study blood heterochronicity on a chip, not just in general, but also in a gender-specific way, a disease-specific way, and a patient-specific way. We are developing this together, going forward.
My last slide is on the mechanism. How do we believe that dilution of plasma in old animals is rejuvenating? What we believe happens is that aging is driven by excessive proteins that at young levels are vital; we cannot really turn off the gene or really indiscriminately remove them with neutralizing antibodies.
They are vital proteins, but when age elevated, they start acting counterproductively and inhibit tissue maintenance and repair as well as inhibiting many other important signal transduction pathways that are needed to build good blood vessels, to have good innervation and a good immune system and so forth. It is a major proteome regulation.
What is shown in this largely blue and largely red comparison is that, surprisingly, interestingly, and, again, counterintuitively, dilution of old systemic milieus in mice and in people results in the elevation of a number of proteins at one week after the procedure in mice, and one month after the procedure in people. There is some downregulation, but there is profound upregulation.
Collectively, what happens is described in this very simplistic, it’s not really a simulation, it’s just an illustration of the process. What happens if there is an age-elevated protein is shown here in red, which maintains itself and inhibits an age-diminished protein shown in green. The dramatic dilution of that protein provides some other feedbacks, which I don’t have time to discuss, which are shown in blue, but, importantly, unleashes and allows expression of the youthful factor.
Dilution of all systemic milieu, in theory, is therapeutic by itself, because you don’t need to add this green useful protein, it did not disappear from your genome, and now when not inhibited is now normally upregulated again and can function. Again, this is published, I don’t have time to discuss it in more detail.
We predict that there will be decaying waves from a single procedure with the overall restoration of gene expression, epigenetics, and proteins to a slightly younger level. The interesting question is, can we then make it slightly even more, a little bit more, a little bit younger at the next procedure, and younger and younger. Is there incremental rejuvenation, instead of returning to the old state after a single procedure, in rounds of procedures?
That summarizes the main points that I would like to discuss. I’m ending my presentation in this diagram view of what really happens, what we believe happens or should happen. The transcriptome and epigenome, since many people ask, did you actually become epigenetically younger?
The answer is, of course, yes. Because based on the central dogma, if you are at a younger level, if you recalibrated all of the levels of the proteins to a younger level, it means that you recalibrated mRNA to a younger level, and it also means that you recalibrated the open and closed chromosomal regions and epigenetics.
Younger cells are not possible without younger proteins, these show that it, in fact, is the case. Younger proteins means that there was younger gene expression, and that means that there was younger epigenetics.
Another point to consider is that our epigenetic status is not really young or old all the time. It is very different from epigenetic settings for liver cells versus hair follicle cells. Cell fate epigenetics are semi stable, but cell behavior or response epigenetics are very, very dynamic.
Shown here on the diagram on the left, it is changing perhaps several times per day in most people, definitely several times per month or per week, and it depends not just on the diet, but also whether it’s raining or shining and if somebody is smoking or not, but also on financial status and how you feel about yourself.
Somebody gave you a hug in the morning and told you that they love you, that releases oxytocin and changes many, many other signal transduction pathways in your cells and changes their epigenetic profile. Epigenetics is dynamic, and there are, of course, known shifts between young and old. Once we show that the proteome was rejuvenated, by default, epigenetics is also younger. That perhaps will apprehend some of the questions from the audience.
I would like to thank everybody in the Conboy laboratory and hope that Dobri can come in again now that the pandemic, hopefully, is winding down. This is our dog, Woofie. Me and Mike and everybody in the lab who contributed to the work I presented.
My conclusion is just one conclusion, it’s written here on top. We are wrapping up the efficacy study with Dr. Dobri Kiprov, who’s clinical director of that pilot study. So hopefully, if I’m invited again to give a talk at this series, that we’ll be able to present the outcomes of this pilot study of heterochronic plasma exchange that is set up for rejuvenation in people.
Thank you so much. Last but not least, I would like to thank many great funding sources who supported this work. Thank you.