Scientists started connecting the circulatory systems of rodents to one another as early as the 1860s, and decades of research since then show that this technique, called parabiosis, can rejuvenate older animals — at least in mice. In the brain specifically, the procedure can make an older animal appear younger, boosting the growth of new cells and blood vessels, and improving memory and other aspects of cognition. Researchers are now trying to understand the cellular and molecular mechanisms underlying its benefits, with the hope that these efforts will translate to new therapies to slow or reverse cognitive decline in humans.
Three new papers are diving into this question by examining how parabiosis alters gene activity in the brain, brain vasculature and other tissues. The studies use single-cell RNA sequencing to measure levels of gene transcription in individual cells, providing a more fine-grained analysis than previously possible. This enables researchers to assess changes in different cell types, an approach that’s especially important in the brain, which contains many types of cells, says Lee Rubin, a neuroscientist at Harvard University and an investigator with the Simons Collaboration on Plasticity and the Aging Brain (SCPAB). Some cell types are very rare, so their contributions could be easily lost in a whole-brain approach.
The findings reveal that young blood seems to restore gene expression patterns to a more youthful state, and that different cell types change in different ways. The results point to potential targets for intervention that could help people retain cognitive function into older age or combat aging-related diseases. “How can you change an old cell into a young cell?” Rubin says. “That’s what we’re asking.”
Building new cells and blood vessels in the brain
Specialists of many kinds have used parabiosis to investigate how factors in the blood might influence health. For example, Rubin says, endocrinologists could do experiments where they connected a diabetic mouse to a non-diabetic mouse. If they observed improvements in the sick animal, they might conclude that some kind of substance in the blood of a non-diabetic mouse could treat the disease. As early as 1959, researchers used parabiosis to show that something circulating in the blood could influence food intake and obesity.
In 2005, after a lull in the experimental use of the procedure, Thomas Rando and collaborators at Stanford University revived it to take a closer look at aging. By connecting a young mouse to an old one in a process called heterochronic parabiosis, they showed that there was functional improvement in the older mouse in a variety of tissues, including its skeletal muscles, heart and liver, a finding borne out in subsequent studies. Other groups have since shown that parabiosis affects brain function as well. Saul Villeda and Tony Wyss-Coray, SCPAB investigators also at Stanford, demonstrated that parabiosis could worsen memory in young animals and improve memory in old animals. Rubin and collaborators found that treating old mice with young blood stimulated neurogenesis in two main areas of the brain where neural stem cells make new neurons: the hippocampus and the subventricular zone. They also observed the regrowth, or revascularization, of blood vessels that deliver glucose and oxygen to brain cells throughout the brains of old mice exposed to young blood. Taken together, the findings suggest that the procedure doesn’t simply slow the aging process, Rubin says. Studies showed rejuvenation or what he likes to call “de-aging”: Old tissues reverted structurally to more closely resemble younger versions, and treated animals behaved more like younger ones, Rubin says. “Overall, they are really surprising and positive results.”
The fact that brains of younger mice given blood from old animals can decline, particularly if the old mice are very old, has helped spur a search for ‘good factors’ and ‘bad factors’ in the blood that can influence the brain. The thinking is that these factors may be made by peripheral tissues or circulating cells and secreted into the blood, and that their production and secretion may be regulated by aging. “The idea has always been that the good factors are more plentiful in young blood [than in old blood],” Rubin says.
Multiple groups have used proteomics to compare young and old blood. The process allows them to identify hundreds of proteins whose levels change with age and might be restored via parabiosis. Scientists have so far characterized at least 10 of these proteins, and each group has a favorite factor to probe, Rubin says. His favorite is GDF11, which may spur neurogeneration and revascularization. Along with Harvard colleagues Amy Wagers and Richard Lee, he helped started a company that is working to get GDF11 into clinical use. Other researchers are doing similar work with other proteins.
Tracking parabiosis’s effects in single cells
In 2019, Rubin and colleagues used single-cell RNA sequencing to analyze how gene expression changes with age in every type of cell in the mouse brain. Building on that work for a newer study, posted as a preprint in January, they conducted the same kind of transcription analysis for every cell type in the brains of 56 mice to examine how old cells responded to treatment with young blood, as well as how young cells responded to old blood.
In both cases, they looked for genes whose expression changed in one direction with aging and in the opposite way with parabiosis, and they correlated those genes with functions, such as senescence, mitochondrial function, or protein quality control. The resulting road map catalogs how gene expression changes throughout the brain and what kinds of effects those changes might have. Rubin’s team has made the massive dataset publicly available for other groups to analyze.
All types of brain cells change with aging and with parabiosis, Rubin says. But they found that endothelial cells were particularly important; these cells show a relatively large number of transcriptional changes with age and after exposure to young blood. Endothelial cells comprise the vasculature where factors in the blood make first contact with the brain, Rubin says, so it makes sense to see a lot of changes in gene activity there. (For more on the brain vascular system, see “New Map of Brain Vasculature Reveals Unexpected Diversity.”)
Rubin hopes the findings will point to new ways to target the interface between blood and the brain, which is vital for brain function. “Could we, starting from our knowledge of the way gene expression changes with aging, use that as the basis for developing a drug that could de-age endothelial cells so they function more like they did in young brains?” he says. “And if so, what diseases could that help?”
Another paper, published in Nature in March, used single-cell RNA sequencing to assess how parabiosis alters transcription across the body. Wyss-Coray and collaborators joined the circulatory systems of pairs of young and old mice — 4-month-old and 19-month-old mice (equivalent to 25- and 65-year-old people) — for five weeks. Researchers analyzed about 50 cell types from 20 organs, including the brain, comparing aging profiles in untreated old mice, old mice that were exposed to young blood, and young mice exposed to old blood.
Researchers identified parabiosis-mediated changes occurring in nearly all cell types, often affecting thousands of genes. Large and consistent changes, with activity going one way with aging and the opposite way with rejuvenation through parabiosis, were identified in multiple cell types. Liver hepatocytes showed the biggest parabiosis-linked reversal of age-related changes in gene expression. The study also found significant changes in endothelial cells, mesenchymal stromal cells and a variety of immune cells. Genes involved in the mitochondrial electron transport chain were particularly responsive to reversal of aging with parabiosis. That pattern held across cell types, says co-author Stephen Quake, president of the Chan Zuckerberg Biohub and a biophysicist at Stanford.
Although the study didn’t focus specifically on the brain, the data include information on brain cells that other researchers can tap into for future analyses, Quake says. He expects the scientific community to be mining the data for years.
The study is part of a series of papers that probe the cellular effects of aging in various cell types, he adds. The researchers previously created a whole-organism cell atlas and a transcriptome analysis of nine mice at various ages; the new paper helps with “understanding the various effects of parabiosis across a broad range of tissues and cell types within the body, especially in the context of aging,” says Róbert Pálovics, a computer scientist at Stanford and co-author of the study.
Aging clocks measure rejuvenation
Parabiosis isn’t the only way to restore youthful function in old cells. Exercise does the same thing, as does caloric restriction. Villeda and collaborators have shown that blood from exercised mice can boost neurogenesis and cognitive function in old animals. Comparing these different techniques could bring new molecular insights into how they trigger rejuvenation, says Rando, director of the Glenn Center for the Biology of Aging at Stanford University School of Medicine in Palo Alto. He has collaborated on research in the last couple of years showing that exercise can rejuvenate stem cells in the brain, muscle and bone marrow.
To compare the age-combating mechanisms of exercise with those of parabiosis, Anne Brunet, co-director of the Glenn Center and an SCPAB investigator, Rando and collaborators first constructed a novel type of aging ‘clock.’ They scrutinized cells in the subventricular zones of 28 mice ranging in age from 3 months, equivalent to a young adult, to 29 months, which is old. Using single-cell RNA sequencing to measure how transcription activity changed with age in different types of brain cells, the researchers identified dozens of genes whose activity varied with age in one direction or the other, as they described in a preprint posted in January. To construct the clock, they used machine learning to select a subset of genes that best predicted age in six types of cells. (Other aging clocks use epigenetic markers in different tissue types rather than gene expression. For more on epigenetic clocks, see “Molecular Clocks Offer New Insight Into Aging.”) One of the most important genes the team identified is involved in inflammation.
Using their clock, the team was able to quantify the magnitude of parabiosis-induced brain rejuvenation in a variety of cell types, highlighting which cell types might be most amenable to interventions and whether interventions that target different cell types could be synergistic. The findings, reported in a preprint in January, also hint at which cell types account for cognitive improvements and other high-level rejuvenation effects, says Eric Sun, a graduate student in computational biology at Stanford and one of the study’s co-authors. As far as he knows, Sun says, it is the first clock to use gene expression at the single-cell level instead of mixing lots of cell types together. The team plans to eventually look at other tissue types, too.
The clock may offer a more efficient way to screen therapeutics — by testing how they alter the clock. “Say you have 1,000 different types of treatments that you think might help in terms of rejuvenating an animal in some way or another,” Sun says. Rather than treating an animal with a drug or other intervention and waiting for its entire life span to assess the effects, researchers can screen compounds by assessing their effects on the clock, he says.
Exercise led to rejuvenation too, the clocks showed, though to a lesser degree than exposure to young blood. When the researchers compared results from parabiosis and exercise, they found that the interventions triggered changes in different genes and cell types. Parabiosis, for example, reduced the activity of certain inflammation-related genes. Exercise, on the other hand, increased the activity of genes that decline with age and are involved in transmembrane transport. Parabiosis mostly rejuvenated activated neural stem cells. Exercise mostly rejuvenated oligodendrocytes. “Overall, we are excited to use these aging clocks as tools to rapidly measure and compare the impact of other interventions in the aged brain,” Brunet wrote in a Twitter thread.
In addition to helping establish a method for measuring aging and age-reversing techniques, the new findings suggest that exercise and parabiosis lead to rejuvenation in distinct ways and that analyses of both could lead to distinct applications, Rando says. “There are a lot of ways that we and others are going to be looking at rejuvenation — with parabiosis, with drugs, with diet, with exercise. How similar are these, and do they ultimately end up in a kind of a final common pathway? What this paper would say is that, at least looking at transcription, they had different ways of restoring youthfulness.”
Together, the results offer new ways to measure how aging affects cells and how those changes can be modified. But the researchers caution that it will take some time to translate those findings into therapies. “There’s a lot of important physiology and biology to work out to understand what’s reasonable to expect to be able to change,” Quake says. “I think we have so much more to learn. It would be premature to say that there’s a clear therapeutic implication.”