Part three of a four-part series.
As the brain ages, its microglial cells turn sluggish in their task of ingesting and degrading toxic products, and the flow of blood through its micro vessels slows. Are there components in the blood that age the brain—and can renew it? At the Zilkha Symposium on Alzheimer’s Disease and Related Disorders, held April 4 at the University of Southern California, Los Angeles, Tony Wyss-Coray of Stanford University, Palo Alto, shared unpublished results from an ongoing study that first characterized a microglial aging phenotype and then partially reversed it with still-unknown factors from the body’s systemic milieu.
The data offered a provocative new twist on the old specter of rejuvenation with young blood. It also reflected the power of heterochronic parabiosis, a surgical protocol of conjoining the blood supply of a young and an old mouse to study complex pathophysiological processes. The new data presented at the Zilkha symposium receives support from a flurry of separate parabiosis papers published on May 4. Led by scientists at the University of California, San Francisco, Harvard Medical School, and other institutions, these three papers demonstrate striking benefits of young blood on cognitive function, synaptic plasticity, neurogenesis, and the cerebral vasculature of old mice. The beneficial effects are not limited to the brain, and the operative factors can be identified, as one study reports that growth differentiation factor GDF11 empowers young blood to bestow regenerative oomph to old muscle.
These two mice, one old one young, live with a shared blood supply. [Image courtesy of Tony Wyss-Coray.]
Aging brings with it not only a decline in cognition but also a smoldering inflammation within the innate immune system. “This low-grade chronic inflammation is bad news,” Terrence Town of the University of Southern California in Los Angeles said at the Zilkha conference. In the brain, this manifests as an abnormal state of that organ’s main resident immune cell, the microglia. For example, expression of the microglial activation marker CD68, a lysosomal protein, rises with age. Electron micrographs of aging brain show microglia with an enlarged, dense nucleus, shriveled Golgi cisternae, few lysosomes, and vesicles jammed with lipufuscin granules. “Aging microglia clearly look abnormal,” Wyss-Coray told the conference audience. They behave abnormally, too, hardly phagocytosing in culture when presented with their usual substrates.
To see whether this is an internal affair of the aging brain or influenced by the periphery, Wyss-Coray returned to a blood-sharing experiment called parabiosis. His lab had previously used it to show that a young systemic environment can essentially rejuvenate neurogenesis and other aspects of the aging brain (see Nov 2009 news story, Mar 2013 news story).
Parabiosis involves suturing the body walls of two mice together such that their capillaries fuse. The mice then live like Siamese twins joined through their blood supply. At the Zilkha conference, Wyss-Coray said that pairing an 18-month-old with a 3-month-old mouse, and letting them live together for five weeks, reversed microglial aging. Microglial activation as measured by CD68 expression was down in the brains of old mice exposed to young blood. In the electron microscope, the old mice’s microglia looked like those of young mice, with a normal-sized, light nucleus, larger Golgi cisternae, more lysosomes, and less lipofuscin.
To the eyes of some scientists at the conference, the nucleus in microglia from old mice looked as if it contained dense chromatin that would allow less protein translation. Wyss-Coray replied that he does not know for sure how the ultrastructural changes in aging microglia relate to gene expression and function.
That said, his lab did compare the microglial transcriptome from old mice paired with other old mice to that from old mice paired with young mice. They saw that blood supplied by a young mouse did indeed largely reverse the gene expression phenotype of microglial aging. This includes age-related increases not only in CD68 and in the complement component C1qB; but also age-related decreases in progranulin, the transcription factor EGR1, and many other expression changes.
In a separate study, Ingenuity Pathway Analysis of gene expression profiles of the aging hippocampus pointed to a synaptic plasticity network anchored by the transcription factors Creb (cAMP response element-binding protein) and EGR1 as being most preserved in rejuvenated old mice. Golgi silver staining spotted more spines on the dendrites of old mice when each had each partnered with a young mouse. A paper published by Wyss-Coray and his former postdoc Saul Villeda and colleagues on May 4 in Nature Medicine reports additional findings to back up the claim of revitalized synapses in heterochronic old mice. These include strengthened long-term potentiation as seen with electrophysiology of cultured hippocampal slices, as well as mechanistic experiments using local expression of dominant-negative Creb and RNA interference of Creb to pinpoint this transcription factor as a hub in the requisite signaling network. “There is some sort of reactivation of a synaptic plasticity network in an old mouse exposed to young blood, ” Wyss-Coray said.
Whether these changes at the molecular and cellular level amount to better function is difficult to assess in parabiotic mice. The pairs run the rotarod together, but rigorous behavior assays are not possible. Instead, the Stanford scientists decided to model parabiosis by transferring young plasma into an old mouse once every three days for three weeks. In this study, old mice injected with plasma from young mice outperformed untreated old mice in the radial arm water maze and a fear-conditioning test. The treated mice also recapitulate other previously shown parabiosis phenotypes, including more neurogenesis, synaptic plasticity, spine density, and less neuroinflammation. The effects are not due to steroid hormones, and happen equally in male and female mice, Wyss-Coray said.
“These results are stunning. Extremely interesting,” commented Berislav Zlokovic of USC.
Are they too good to be true? “That is what some reviewers said,” Wyss-Coray replied dryly. The latest findings of this line of research are not yet published, but previous work appeared in 2009 and has been awaiting independent replication. Villeda has started his own lab at UCSF, where new students have reproduced the findings. Beyond that, few other labs have independently replicated them yet.
That may be beginning to change with a new paper, released also on May 4, in Science magazine. In this study, researchers led by Lee Rubin at the Harvard Stem Cell Institute in Cambridge, Massachusetts, report that young blood reinvigorated blood vessels of the neurogenic niche in the brain of old mice. In heterochronic old mice, the volume of cerebral blood vessels almost doubled. They formed new branches and allowed more blood to flow through (see movie below). This, in turn, supported a robust increase in the number of neural stem cells in the old mice’s sub-ventricular zone, a brain area that is a source of new neurons throughout life but runs dry in aging (see news story on Science Now).
Blood flow in an old mouse brain. [Courtesy of John Chen and Greg Wojtkiewicz]
Blood flow in a rejuvenated old mouse brain. [Courtesy of John Chen and Greg Wojtkiewicz]
This paper confirms Wyss-Coray’s 2009 findings that factors in young blood stimulate neurogenesis in the old brain and that factors in old blood slow neurogenesis in the young brain. Rubin’s paper refines Wyss-Coray’s by reporting that it is not until mice are truly old—in this case 21 months—that their blood impairs neurogenesis of young mice. Blood from 15-month-old mice did not, suggesting that it is only during aging that these negative factors accumulate in the blood.
These new papers aside, why were scientists at large slow to catch on to the potential of parabiosis for the study of brain aging? It may be partly because parabiosis has fallen out of favor over the past two decades and appears only now to experience a small revival. Greek for “living alongside,” this experimental system has a storied history. Used widely in physiology and endocrinology research during the first 70 years of the 20th century, parabiosis advanced the fields of growth and sex hormones and set the stage for the discovery of parathyroid hypertensive factor. Parabiosis showed the presence of the satiety factor that was later called leptin, a discovery that garnered the 2010 Albert Lasker Basic Medical Research Award (Coleman 2010). In 1972, parabiosis showed that old rats lived longer and were more vigorous when conjoined to young rats (Ludwig et al., 1972. Incidentally, the “trans” in this citation stands for ‘Transactions,” not "Transylvania"). Alas, the “creep factor” of creating a surgical bondage in the service of science may have set animal care committees against the technique, Wyss-Coray said, and it faded from use.
In a recent review article arguing for a return of parabiosis to study the pathophysiology of age-related disease, Wyss-Coray writes that paired mice fare better than many other animal models exposed to pathogens, traumatic injuries, cancer, or debilitating mutations (Eggel and Wyss-Coray, 2014). In Los Angeles, he noted unpublished recovery data showing that the pairs resumed grooming and nesting, and lived a full lifespan.
Wyss-Coray’s lab initially learned parabiosis from fellow Stanford scientist Tom Rando, who used it to stimulate regeneration in liver and muscle (Conboy et al., 2005). Since then, heterochronic parabiosis has boosted recovery in models of multiple sclerosis and heart failure due to weakening cardiac muscle (Ruckh et al., 2012; Loffredo et al., 2013). Indeed, this last study, by the groups of Lee Rubin and Amy Wagers, also at the Harvard Stem Cell Institute, gave rise to the third paper published on May 4. In it, researchers led by Wagers report that the circulatory protein GDF11 rejuvenates not only heart but also skeletal muscle. The scientists first characterized how heterochronic parabiosis restored in old mice the muscle satellite cells that promote muscle healing, and then went on to show that daily injection of recombinant GDF11 generated much the same phenotype. The new paper by Rubin et al. on the effects of young blood on the neurovascular niche of aged mice also showed that daily injection of GDF11 alone pulled off about half the beneficial effect on the brain’s capillaries and neurogenesis as that noted for whole young blood in parabiosis.
In the view of other scientists at the Zilkha conference, the apparent success of the plasma transfer protocol and even targeted injection of candidate humoral factors is likely to prompt more laboratories to take up parabiosis.
In a press release issued by Harvard University, Rubin is quoted extensively. “We think an effect of GDF11 is the improved vascularity and blood flow, which is associated with increased neurogenesis. [This] should have more widespread effects on brain function,” he said. "We do think that, at least in principle, there will be a way to reverse some of the cognitive decline that takes place during aging. It isn't out of question that GDF11, or a drug developed from it, might be capable of slowing some of the cognitive defects associated with Alzheimer's disease."
Rubin is further quoted as saying that a future treatment for Alzheimer’s might be a combination of a therapeutic that reduces plaques and tangles with a potential cognition enhancer like GDF11.—Gabrielle Strobel
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- Eggel A, Wyss-Coray T. A revival of parabiosis in biomedical research. Swiss Med Wkly. 2014 Feb 4;144:w13914. PubMed.
- Conboy IM, Conboy MJ, Wagers AJ, Girma ER, Weissman IL, Rando TA. Rejuvenation of aged progenitor cells by exposure to a young systemic environment. Nature. 2005 Feb 17;433(7027):760-4. PubMed.
- Ruckh JM, Zhao JW, Shadrach JL, van Wijngaarden P, Rao TN, Wagers AJ, Franklin RJ. Rejuvenation of regeneration in the aging central nervous system. Cell Stem Cell. 2012 Jan 6;10(1):96-103. PubMed.
- Loffredo FS, Steinhauser ML, Jay SM, Gannon J, Pancoast JR, Yalamanchi P, Sinha M, Dall'Osso C, Khong D, Shadrach JL, Miller CM, Singer BS, Stewart A, Psychogios N, Gerszten RE, Hartigan AJ, Kim MJ, Serwold T, Wagers AJ, Lee RT. Growth differentiation factor 11 is a circulating factor that reverses age-related cardiac hypertrophy. Cell. 2013 May 9;153(4):828-39. PubMed.
No Available Further Reading
- Villeda SA, Plambeck KE, Middeldorp J, Castellano JM, Mosher KI, Luo J, Smith LK, Bieri G, Lin K, Berdnik D, Wabl R, Udeochu J, Wheatley EG, Zou B, Simmons DA, Xie XS, Longo FM, Wyss-Coray T. Young blood reverses age-related impairments in cognitive function and synaptic plasticity in mice. Nat Med. 2014 Jun;20(6):659-63. Epub 2014 May 4 PubMed.
- Katsimpardi L, Litterman NK, Schein PA, Miller CM, Loffredo FS, Wojtkiewicz GR, Chen JW, Lee RT, Wagers AJ, Rubin LL. Vascular and neurogenic rejuvenation of the aging mouse brain by young systemic factors. Science. 2014 May 9;344(6184):630-4. Epub 2014 May 5 PubMed.