In 2005, University of California, Berkeley, researchers made the surprising discovery that making conjoined twins out of young and old mice – such that they share blood and organs – can rejuvenate tissues and reverse the signs of aging in the old mice.
The finding sparked a flurry of research into whether a youngster’s blood might contain special proteins or molecules that could serve as a “fountain of youth” for mice and humans alike.
But a new study by the same team shows that similar age-reversing effects can be achieved by simply diluting the blood plasma of old mice – no young blood needed.
In the study, the team found that replacing half of the blood plasma of old mice with a mixture of saline and albumin – where the albumin simply replaces protein that was lost when the original blood plasma was removed – has the same or stronger rejuvenation effects on the brain, liver and muscle than pairing with young mice or young blood exchange.
Performing the same procedure on young mice had no detrimental effects on their health.
This discovery shifts the dominant model of rejuvenation away from young blood and toward the benefits of removing age-elevated, and potentially harmful, factors in old blood.
“There are two main interpretations of our original experiments: The first is that, in the mouse joining experiments, rejuvenation was due to young blood and young proteins or factors that become diminished with aging, but an equally possible alternative is that, with age, you have an elevation of certain proteins in the blood that become detrimental, and these were removed or neutralized by the young partners,” said Irina Conboy, a professor of bioengineering at UC Berkeley who is the first author of the 2005 mouse-joining paper and senior author of the new study.
“As our science shows, the second interpretation turns out to be correct. Young blood or factors are not needed for the rejuvenating effect; dilution of old blood is sufficient.”
In humans, the composition of blood plasma can be altered in a clinical procedure called therapeutic plasma exchange, or plasmapheresis, which is currently FDA-approved in the U.S. for treating a variety of autoimmune diseases.
The research team is currently finalizing clinical trials to determine if a modified plasma exchange in humans could be used to improve the overall health of older people and to treat age-associated diseases that include muscle wasting, neuro-degeneration, Type 2 diabetes and immune deregulation.
“I think it will take some time for people to really give up the idea that that young plasma contains rejuvenation molecules, or silver bullets, for aging,” said Dobri Kiprov, a medical director of Apheresis Care Group and a co-author of the paper.
“I hope our results open the door for further research into using plasma exchange — not just for aging, but also for immunomodulation.”
The study appears online in the journal Aging.
A molecular ‘reset’ button
In the early 2000s, Conboy and her husband and research partner Michael Conboy, a senior researcher and lecturer in the Department of Bioengineering at UC Berkeley and co-author of the new study, had a hunch that our body’s ability to regenerate damaged tissue remains with us into old age in the form of stem cells, but that somehow these cells get turned off through changes in our biochemistry as we age.
“We had the idea that aging might be really more dynamic than people think,” Conboy said. “We thought that it could be caused by transient and very reversible declines in regeneration, such that, even if somebody is very old, the capacity to build new tissues in organs could be restored to young levels by basically replacing the broken cells and tissues with healthy ones, and that this capacity is regulated through specific chemicals which change with age in ways that become counterproductive.”
After the Conboys published their groundbreaking 2005 work, showing that making conjoined twins from the old mouse and a young mouse reversed many signs of aging in the older mouse, many researchers seized on the idea that specific proteins in young blood could be the key to unlocking the body’s latent regeneration abilities.
However, in the original report, and in a more recent study, when blood was exchanged between young and old animals without physically joining them, young animals showed signs of aging. These results indicated that that young blood circulating through young veins could not compete with old blood.
As a result, the Conboys pursued the idea that a buildup of certain proteins with age is the main inhibitor of tissue maintenance and repair, and that diluting these proteins with blood exchange could also be the mechanism behind the original results.
If true, this would suggest an alternative, safer path to successful clinical intervention: Instead of adding proteins from young blood, which could do harm to a patient, the dilution of age-elevated proteins could be therapeutic, while also allowing for the increase of young proteins by removing factors that could suppress them.
To test this hypothesis, the Conboys and their colleagues came up with the idea of performing “neutral” blood exchange. Instead of exchanging the blood of a mouse with that of a younger or an older animal, they would simply dilute the blood plasma by swapping out part of the animal’s blood plasma with a solution containing plasma’s most basic ingredients: saline and a protein called albumin.
The albumin included in the solution simply replenished this abundant protein, which is needed for overall biophysical and biochemical blood health and was lost when half the plasma was removed.
“We thought, ‘What if we had some neutral age blood, some blood that was not young or not old?’” said Michael Conboy.
“We’ll do the exchange with that, and see if it still improves the old animal. That would mean that by diluting the bad stuff in the old blood, it made the animal better. And if the young animal got worse, then that would mean that that diluting the good stuff in the young animal made the young animal worse.”
After finding that the neutral blood exchange significantly improved the health of old mice, the team conducted a proteomic analysis of the blood plasma of the animals to find out how the proteins in their blood changed following the procedure.
The researchers performed a similar analysis on blood plasma from humans who had undergone therapeutic plasma exchange.
They found that the plasma exchange process acts almost like a molecular reset button, lowering the concentrations of a number of pro-inflammatory proteins that become elevated with age, while allowing more beneficial proteins, like those that promote vascularization, to rebound in large numbers.
“A few of these proteins are of particular interest, and in the future, we may look at them as additional therapeutic and drug candidates,” Conboy said. “But I would warn against silver bullets.
It is very unlikely that aging could be reversed by changes in any one protein. In our experiment, we found that we can do one procedure that is relatively simple and FDA-approved, yet it simultaneously changed levels of numerous proteins in the right direction.”
Therapeutic plasma exchange in humans lasts about two to three hours and comes with no or mild side effects, said Kiprov, who uses the procedure in his clinical practice.
The research team is about to conduct clinical trials to better understand how therapeutic blood exchange might best be applied to treating human ailments of aging.
Co-authors of the paper include Melod Mehdipour, Colin Skinner, Nathan Wong, Michael Lieb, Chao Liu, Jessy Etienne and Cameron Kato of UC Berkeley.
Parabiosis is a surgical technique that involves joining the circulatory system of two animals such that they continuously exchange blood and other circulating factors. About a decade ago, this method was used to test whether the age of one animal has effects on the health of its partner through heterochronic parabiosis, where a young mouse shares it circulatory system with an old mouse.
Strikingly, muscle stem cells and liver cells from the young mouse functioned less well, while the same cells from the old mouse showed molecular and functional evidence for rejuvenation (Conboy et al., 2005).
Since then, similar effects have been demonstrated in other tissues including spinal cord (Ruckh et al., 2012), heart (Loffredo et al., 2013), and brain (Villeda et al., 2011). Recently, this work has been extended by the finding that injecting plasma from young mice is sufficient to enhance cognitive function and synaptic plasticity in aged mice (Villeda et al., 2014), and the identification of two molecules as key mediators of the beneficial and negative consequences from heterochronic parabiosis (Figure 1), Growth Differentiation Factor 11 (GDF11) (Katsimpardi et al., 2014; Sinha et al., 2014) and C-C motif chemokine 11 (CCL11) (Villeda et al., 2011).
GDF11, a member of the TGF-β superfamily, declines in blood with age (Loffredo et al., 2013), and restoration of youthful levels of GDF11 is sufficient to enhance stem cell and tissue function in heart (Loffredo et al., 2013).
In contrast, blood levels of CCL11 increase with age, and this increase appears to contribute to the decline in neurogenesis and function of neural stem cells in the hippocampus (Villeda et al., 2011). Now, two new studies have found that GDF11 also has beneficial effects on skeletal muscle, the sub-ventricular nuclei and the hippocampus.
Supplementation with GDF11 alone restored skeletal muscle strength, physical endurance, and regeneration following injury in aged mice (Sinha et al., 2014). Similarly, old mice treated with GDF11 had improved olfactory perception, brain vascularization, and neural stem cell function, which could translate into increased protection of the nervous system against age related challenges (Katsimpardi et al., 2014).
Conversely, injecting CCL11 impaired learning and memory in young mice, likely by reducing neurogenesis in the hippocampus (Villeda et al., 2011). A CCL11-neutralizing antibody abrogated the negative effects of CCL11 treatment in young mice, although it was not reported whether the CCL11-neutralizing antibody alone could improve function in aged mice.
Another recent study suggests that the fruit fly homolog of GDF11, myogliannin, is secreted by muscle to regulate aging in that organism (Demontis et al., 2014). Demontis and colleagues found that overexpression of the Mnt transcription factor specifically in muscle was sufficient to attenuate age-associated declines in climbing ability and extend lifespan. Interestingly, it also caused changes in nucleolar structure of both muscle and adipocyte cells, suggesting that muscle Mnt has both cell autonomous and cell non-autonomous effects. Myogliannin was identified as a secreted factor induced by Mnt that mediates these effects, and overexpression of myogliannin specifically in muscle extends lifespan.
Although it remains unclear whether myogliannin functions similarly to GDF11 (myogliannin is also homologous to myostatin and appears to function in some ways similarly to myostatin), the observation that both myogliannin and GDF11 are secreted factors that modulate aging-related phenotypes in flies and mice is intriguing.
Recent advances in aging research have led to the identification of a small but growing number of interventions that enhance longevity and promote healthy aging. For example, dietary restriction or treatment with the mTOR inhibitor rapamycin have both been found to increase lifespan in yeast, nematodes, fruit flies, and mice.
Such interventions, if they can be successfully translated to people, have the potential to dramatically impact human health by simultaneously delaying the onset and progression of multiple age-related disorders (Kaeberlein, 2013). As such, the discovery of systemic factors that appear to modulate aging, such as GDF11 and CCL11, has potentially profound therapeutic implications.
If similar mechanisms occur in people, then providing elderly individuals with plasma from young individuals, or even more specifically with GDF11 or a CCL11-neutralizing antibody, may lead to improved function of multiple organ systems. Given that these approaches are essentially restoring levels of our bodies’ own molecules toward a more youthful state, they may prove less prone to side effects compared to pharmacological interventions that slow aging, such as rapamycin.
On the other hand, altering the abundance of specific molecules in the context of an aged system may have unanticipated effects that are different from the young state. In this regard, it is rather surprising that it hasn’t yet been reported whether longer-term heterochronic parabiosis, or more specific treatments in aged animals such as young plasma or GDF11, does in fact significantly improve healthspan or lifespan of mice.
This is important, both for evaluating the effects of such interventions in the context of the aging animals, as well as for understanding whether there are any substantial negative side effects associated with such treatments.
A related question is whether continuous treatment is required to fully rejuvenate tissues of aged mice, or perhaps a transient exposure to these factors might be sufficient. For example, injecting young plasma into aged mice eight times over a period of twenty-four days resulted in significant improvements in hippocampal-dependent learning and memory immediately following treatment (Villeda et al., 2014), but it is unclear whether these benefits persist or are rapidly lost once treatment is stopped.
Finally, it remains to be ascertained whether these systemic factors act within well-known aging pathways, such as mTOR and growth hormone/insulin-IGF-1 signaling, or if they are effectors of novel aging pathways. Indeed, the factors regulating the expression of GDF11 and CCL11, the tissues involved in their production, their mechanism(s) of action, and why their expression changes during aging remain to be determined.
These discoveries set the stage for interesting times, as many remaining questions begin to be answered and the translational potential is explored. It seems likely that GDF11 and CCL11 are only the first two in a series of circulating molecules that will be found to influence aging of different tissues.
Whether these are the most important or most potent molecules remains to be seen. Future studies in this area will likely bring forth new and exciting knowledge about the dynamics of aging and novel approaches to regenerative medicine.