Robust rejuvenation through exosomes

Cross-posted from my ScienceBlog on aging

A study out of Nanjing University last month brings exosome rejuvenation to the mainstream of researchers with broad new evidence and some speculations on mechanisms. The prominent publication in Nature Aging corroborates and greatly expands results from Harold Katcher’s Mumbai lab. Massive infusions of exosomes from young mice into old improve cognition, endurance, fertility, energy metabolism, heart function, immune function, bone density, cell senescence, and maximum lifespan.

History and context

This current research grew from the Stanford lab of Tom Rando 20 years ago. An old mouse and a young mouse were sewed together so they shared a common blood supply. The old mouse showed signs of rejuvenation and the young mouse showed signs of accelerated aging. Irina and Mike Conboy graduated and set up their own lab at Berkeley, where they demonstrated that it was not blood cells but something in the plasma that was responsible for the effect. 

Aging is not something that “happens” to cells. It is centrally orchestrated and information about age is transmitted through the bloodstream. Aging at the cell level responds to signals in the blood, even to the extent that old cells can become young in a “young environment”.

The Conboys worked on removing old signaling from the blood. In cooperation with Dobri Kiprov, there is an ongoing human trial, simply removing blood plasma (but not the red or white blood cells) as a therapy.

Twelve years ago, James P Watson whispered to me, “exosomes”, but I was too distracted to listen. 

Seven years ago, Harold Katcher made an inspired guess and quietly conducted research in Mumbai, infusing rats with a blood plasma fraction he would only identify as “elixir” or “E5”. In 2020, his team announced that they had turned two-year-old rats to one-year-old rats according to the Horvath multi-species clock. I wrote about it as a “breakthrough”.

From 2020-23, research proceeded slowly because Harold’s business partner, Akshay Sanghavi, had trouble raising funds for industrial scale extraction of “E5”, and no one else knew what was in it.

Then, last summer, a research group at Smidt Heart Inst in Los Angeles announced promising results rejuvenating rat hearts with exosomes from young rats. Akshay consented to reveal the secret that E5 was exosomes.

What is an exosome? All cells release tiny information packages, wrapped in fats like a lipid nanoparticle. They contain DNA, RNA, proteins, as well as lipids, that communicate both within the body and through the air to other living things.

Would the phenotypic and epigenomic rejuvenation that Harold and Akshay observed lead to dramatically longer lifespans? We had data from only eight rats (all female), and results are not as consistent as we might hope. Median lifespan increased ~20%, depending what you use for a baseline, but curiously the maximum lifespan seems to be extended by 60% or more.

The current paper adds lifespan data from only eight more rats (all male), but the range of metabolic and performance tests is greatly expanded.

Introduction

I predict that this new paper will bring exosome therapy into the mainstream. It begins inauspiciously.

“Aging is an inevitable, time-dependent process that eventually limits the capacity of cells to maintain efficient homeostasis and repair mechanisms…” 

“Inevitable”? If they really thought so, then they wouldn’t be doing this research. My guess is that journal editors demanded this kind of language, both because I’ve experienced such editor arrogance myself, and because Chinese researchers are less hypnotized by the Selfish Gene ideology than biologists in the West. 

They continue, “Mechanistically, the aging process is predominantly attributed to the progressive accumulation of stochastic damages in cells, organelles and macromolecules.” This is pure dogma. Despite this disclaimer, the method of their experiment is not to address stochastic damage in cells, but to adjust signaling in the organism as a whole.

The fact that aging can be reversed with system-level signaling is a sign that it isn’t just “entropy”, that the body is in control, and, of course, that aging is not “cell autonomous” but centrally coordinated. Harold understands this, and it has been central to his key insights. But the mainstream theorists can’t believe that natural selection would be so perverse, and they remain stuck in older paradigms. Perhaps these Chinese authors were compelled to make obeisance to old British dogmas as a price for being published in Nature. The paper was held up in peer review for an inexcusable 27 months.

Results

Old mice, starting in late middle age = 20 months, were given weekly intravenous infusions of exosomes from young mice for as long as they continued to live.

Median lifespan was increased 12%, and maximum lifespan increased 20%. This reminds us of Harold’s exosome-treated rats, one of which lived much longer than the other seven. Usually we think that median lifespan is more malleable than maximum lifespan. For example, exercise increases median and mean lifespan, but not maximum lifespan. Here we find the opposite. More about this further down this column.

• Exosome-treated mice scored much lower on a frailty scale than untreated mice, but were not fully restored to their young, “zero frailty” state.

• Sperm counts and sperm motility were restored to young values, testosterone levels increased, and treated mice were as fertile as young mice. (all specimens were male)

• Metabolic rate (oxygen consumption) was restored about halfway to youthful values. This reflected activity rates, which were improved but not as active as young mice.

• Ejection fraction and other measures of heart performance were improved, but not to the level of young mice.

• Parts of the brain that atrophy in old mice regrew toward youthful volume.

• Memory, measured by a water maze test, was restored to youthful levels in exosome treated mice.

• Treadmill endurance came back almost to youthful levels.

• Treated mice had lower markers for cell senescence. 

• Glycation is a kind of molecular damage that increases in old mice and old humans. Glycation was observed to decrease in treated mice. 

• Treated mice restored bone density lost to osteoporosis.

• The Nanjing group did not report results from the Horvath epigenetic clock for rodents, however they did their own analysis of protein expression in various tissues, and showed that the proteomes of treated mice reverted toward younger profiles.  

• As a control, young mice were infused with exosomes from old mice. Both their endurance and their memories were impaired. Exosomes from young mice have a rejuvenating effect and exosomes from old mice accelerate aging.

Mitochondria are electrochemical energy sources, and cells typically have hundreds to thousands of mitochondria. Mitochondria can reproduce and renew inside a single cell, but populations of mitochondria decline with age. We literally have less energy as we age. The Nanjing group documented restoration of mitochondrial populations in treated mice, and they assigned special significance to this, speculating that this could be a primary source of other rejuvenation effects. The title of the paper singles out mitochondria as a mechanism. Reading through the rationale, I don’t understand why the authors consider mitochondria to be the root of the exosomes’ anti-aging benefits.

My personal belief is that 20% increase in maximum lifespan — impressive as it is — is just the beginning. Exosome technology has not even begun to be optimized.

Dosage

What is the dosage of infused exosomes compared to the innate exosomes resident in the blood of the mice? The article does not offer this information directly; neither do we have specifications from Harold’s experiments. The best I was able to do was to make an estimate from the size of mice and from the provided information “200 microlitres in a weekly dose” and “1.8 micrograms total protein per microlitre”. From this, I guestimated that the infused exosomes are sufficient in quantity to overwhelm the innate exosomes, perhaps 10x the quantity. From some of Harold’s offhanded comments, I had roughly the same impression.

This large quantity underscores the difficulty of sourcing if this technology is to become widely available to humans. We might need 100 piglets per year per human patient, overwhelming the existing market for pork. Or we might need to develop in vitro technologies for growing stem cells that secrete young exosomes. Or we might be able to target a much smaller dose of exosomes to a part of the body that keeps track of time — my candidate is the hypothalamus. 

Or, if it turns out that the activity is due to specific RNAs, we might synthesize them. See below.

Mechanism

“Given the above-observed phenotypes, it remains unclear which component is responsible for the rejuvenating effects of young plasma sEVs.” [sEVs are “small extracellular vessicles”, another name for exosomes.]

The assumption implicit in this statement is that there is just one chemical species in young-derived exosomes that is responsible for all the benefits. When will we absorb the message that living organisms are not like human-designed machines? The association of one protein with one function is vanishingly rare. Almost always, individual functions are not performed by individual chemicals. Rather, there are overlapping complexes of chemicals responsible for what we regard as a single function. Every chemical has multiple functions, and every function is accomplished by multiple chemicals in concert.

Thus, I think it likely that no reduction of the exosome’s complexity will be discovered, and hence there will be no patented single-bullet solutions to aging forthcoming from these experiments. But again, read on…

Exosomes include proteins, RNAs, DNAs, and lipids. All these molecules carry information, and it may be that they all work together to whisper “young” when they are taken up by an aged cell. 

The Nanjing authors nevertheless guessed that there might be a few RNA species that did all the heavy lifting and went looking for them with the tools of data mining that have become fashionable among molecular biologists. 

They report limited success in identifying crucial RNA species. “Thus, miR-144-3p, miR-149-5p and miR-455-3p encapsulated in young plasma sEVs are the key rejuvenating miRNAs and have the potential to stimulate PGC-1α expression and improve mitochondrial energetic metabolism.” This claim is in fact an overstatement. What they succeeded in proving was that blocking all three of these RNAs with RNA interference was sufficient to negate the benefits of the therapy. 

So, yes, the benefit seems to require at least one of these three RNAs, but it also may require a great many more RNAs that are “essential”. And it may be that if any one RNA is missing, others can substitute. After all, the reason that biology is evolved to use these complex, multipronged chemical mechanisms is that they are robust to disruption, much more so than human-designed machines which, typically, can fail catastrophically if a single part is broken. 

The study also includes investigation of the role of RNAs in the age-accelerating effect of old-derived exosomes. They report several RNAs from old-derived exosomes that are potential culprits: “Therefore, miR-29a-3p, miR-29c-3p and miR-34a-5p in aged plasma sEVs are the key pro-aging miRNAs with an entirely opposite function to miR-144-3p, miR-149-5p and miR-455-3p encapsulated in young plasma sEVs.” They use RNA interference to block these specific RNAs, and find that the old exosomes lose their detrimental effect. 

Questions for research

This work with specific RNAs that are pro-aging and anti-aging suggests that RNA therapies might be devised that bypass the need for animal-derived exosomes. These would be patentable, manufactured products which might attract capital investment. Creating a viable therapy from a combination of RNA and RNA interference products, presumably packaged in lipid nanoparticles, could be an attractive research direction for pharma companies.

As a first step, it would be useful to analyze RNAs of exosomes derived from young and old human donors and to quantify the differences.

Determining the natural source of the exosomes could be crucial. Are exosomes that carry age information generated from a “clock region” in the body, perhaps in the hypothalamus? Or are the most relevant exosomes created by many tissues, all over the body, so that we can think of the whole body keeping track of age and coordinating age information system-wide with exosomes in the blood plasma?

Can the treatment be made more effective if “old” exosomes are removed at the same time that “young” exosomes are added?

Certainly exosomes carry a variety of information unrelated to age. Are there specialized exosomes that carry age information? Or is age information one component of the information in all exosomes?

The authors suggest that a useful step would be labeling donor exosomes with radioisotopes so that they can be traced in the mouse who receives them, and tissues can be identified where they are absorbed preferentially. I would add to this the suggestion that different mice might absorb the exosomes in different tissues, and this might be correlated to the variable effect.

Is it feasible to tag different source organs, so we can determine where the age-relevant exosomes are produced?

Response to exosome therapy as currently conceived seems to be highly variable. Animals who respond best live a lot longer, while others die within the expected time frame. The limited data from Harold and from Nanjing both support this. 

This makes me think of the mRNA vaccine technology introduced for COVID. Exosomes are lipid nanoparticles, and lipid nanoparticles are the delivery mechanism of the mRNA products. The present article presents evidence that RNAs are at least part of the mechanism of action. 

Some people who took the mRNA shots got protection from COVID, while others got heart disease and still others suffered neurological damage. I think that LNPs and exosomes go everywhere in the body, and their effect depends on where they are taken up.

There is speculation that in response to certain exosomes, cells can sometimes decide, “this is an important message that I want to pass on”. Cells can create similar exosomes and amplify the message, like a virus or a “tweet that goes viral”. This is speculation, but not far from the edge of exosome behavior that has been established. Cells undergoing apoptosis emit exosomes that can signal other cells into apoptosis. [Apoptosis is cell suicide.] It is reasonable to regard viruses as exosomes that have gone rogue and evolved to maximize their own reproduction — to hell with intercellular messaging. It is likely that exosomes are an essential vehicle of cancer metastasis.

Exosomes for the Hypothalamus?

I have written about suggestions by Dongsheng Cai and Claudia Cavadas that there is an aging clock in the hypothalamus. We know that exosomes can sometimes cross the blood brain barrier. I wonder if the rejuvenating effect of exosomes is strongest if they reset an age clock in the hypothalamus. It is within current experimental technique to target exosomes to the brain and ask this question experimentally. 

Whether or not the hypothalamus plays a central role, it will be interesting to discover whether animals rejuvenated with exosomes can transition toward producing younger exosomes, so that the rejuvenation becomes self-sustaining.

Directions for translational medicine

One of the (many) promising features of exosome intervention is that it seems to compress morbidity. In other words, healthspan is extended more than lifespan. Whatever our feelings about lifespan, we are all looking to remain healthier, longer.

There remains much to be done before exosome therapy can be widely distributed to humans. First on my list would be a factory scale source of young exosomes. Harold and Akshay have suggested that blood from the millions of young pigs slaughtered each year for meat could be a feedstock. Before this can be realized,

• We need to confirm that exosomes from pigs are able to rejuvenate humans.

• We should check that factory-farmed pigs are not impaired in the quality of their exosomes.

• We need industrial scale separation and refinement techniques for extracting exosomes efficiently from blood.

Summary

These results corroborate a theoretical framework in which the age state of the body is communicated to somatic cells, which obediently can become old or young in response. Exosomes seem to be a prime candidate for the communication mechanism. Their lipid coatings enable them to slip easily through cell membranes, and their cargoes include a diverse array of active signal molecules. 

It is my hope that with this new paper, the concept of exosome rejuvenation explodes into the mainstream of anti-aging medicine. There is much work to be done before we have treatments for humans available at scale.

It is extra-promising that healthspan seems to be universally improved, even in animals where lifespan does not change. Another auspicious sign is that maximum lifespan is augmented more than median lifespan in this study as well as Harold’s experiment.

Rejuvenation via exosomes appears to be quite variable across individual mice, despite the fact that these mice are inbred to be genetically identical. This suggests that a lot can be learned from studies comparing mice that respond well to mice that don’t respond at all. 

If specific RNAs can be identified that do the heavy lifting, then these might be synthesized artificially. If, on the other hand, the whole package of DNA, RNA, protein, and lipid molecules in an exosome is found to work together, then we will need industrial-scale extraction from animal sources.

If we are really lucky, then targeting exosome therapy to the hypothalamus could greatly reduce the quantity per treatment and the cost of the treatment.