20 November 2009. Bram Stoker would have loved it. One hundred twelve years after the Irish author penned Dracula, his bone-chilling masterpiece, scientists at last month’s Society for Neuroscience conference in Chicago confirmed the vampire principle, whereby young blood keeps an aging organism vigorous. Of course, the qualifications that must follow such a breezy analogy are manifold. To begin with, the research involved not fair maidens and an ancient count but young and old mice, none of them evil. But in essence, scientists did report that admixing young blood to old rejuvenated the aging brain’s otherwise flagging output of newly generated neurons. The scientists identified some of the molecular factors, to boot. Presented October 19 in a talk by Saul Villeda, a Ph.D. student in the laboratory of Tony Wyss-Coray at Stanford University, the research features a combination of the lab’s parabiosis (i.e., blood-mixing) experiments with proteomics and follow-up studies in vitro and in vivo. The data presented to date are about aging, not Alzheimer disease, but the scientists are actively studying what their findings could mean for this neurodegenerative disease.
Aging remains the biggest risk factor for AD, and it can be viewed as a shift over time in the body’s balance of regenerative capacity versus degeneration, Villeda said. The immune system, in particular, changes with aging. Indeed, many of the signaling proteins active in aging, in stressed neurons, and in activated glial cells are proteins that were originally named by immunologists but function in the brain, as well. “It has been shown that as we age, and in AD, inflammatory proteins in blood correlate with degenerative brain disease in certain ways. The drive behind my work is that I am interested particularly in the regenerative aspect of blood-derived factors, and I view neural stem cells as a readout of that,” Villeda said.
The importance of adult neurogenesis is not fully established, but scientists increasingly believe the new neurons are functionally relevant to olfaction and perhaps learning (e.g., Kokovay et al., 2008). Adult neurogenesis in young and old animals also responds to external stimuli such as exercise, which increases blood supply to the brain and benefits learning and memory.
Wyss-Coray’s group has found, as have published studies before, that adult neurogenesis in the hippocampal subgranular zone of mice dwindles as they age, to where it hovers near zero by the time they are two years old. To pinpoint peripheral factors that might correlate with the age-related decline in neurogenesis, the Wyss-Coray lab conducted a proteomics study of blood from healthy humans at different ages and then tested if the protein signature that came up in this experiment was able to predict age in a similar proteomics run in mice. (As in an earlier proteomics study [Ray et al., 2007; Britschgi et al., 2009], the Stanford scientists restricted their comparison to proteins involved in what they call the “communicome,” i.e., the several hundred secreted proteins known to be involved in signaling among cells of the immune system. This narrows by about a factor of 100 to 1,000, respectively, the challenge of drawing biological meaning out of changes in the entire transcriptome or proteome.) This new proteomics experiment generated a short list of some 12 plasma proteins, of which eotaxin and MCP-1 looked most intriguing, Villeda said.
To take these correlative clues to a functional level, the researchers availed themselves of a technique called parabiosis, where a scientist opens the peritoneum of two mice and sutures them together. This is not vascular surgery; rather, the capillary beds of both mice fuse as their tissue heals. Jian Luo in the Wyss-Coray lab in this way conjoined several dozen pairs of one two-month old and one 18-month old mice. The pairs then lived as unequal Siamese twins of sorts (one small and fluffy, one large and a bit scruffy). They did that for two months while old blood mixed with young. Parabiosis is not new in science (e.g., Mildner et al., 2007), and indeed has inspired artists over the years (e.g., The Two Fridas [.pdf]; Parabiosis). The new aspect here is the combination of young, old, and a neurogenesis readout.
When the mice were four and 20 months old, respectively, Villeda assessed several readouts in the brain. The scientists were startled to find that the young mice exposed to old blood had a drop in neurogenesis as measured by doublecortin-positive cells in the hippocampus, whereas the old mice exposed to young blood enjoyed a threefold boost in their neurogenesis. The newly differentiated neurons in these “Dracula” mice grew longer neurites, too. Also in the hippocampus, the 20-month-old mice were spared the increase in activated microglia that is typically seen in aging mice, whereas the four-month-old mice did show that increase.
Next, the scientists probed whether the candidate factors eotaxin and MCP-1 might play a role in this phenomenon. The concentration of both proteins went up in the brain of young mice connected to old blood, as indeed it does in normally aging mice. These proteins also increase with age in human plasma and CSF, Villeda noted. When tested in vitro, eotaxin slowed stem cell proliferation and neurosphere growth. Similar results came out of in-vivo imaging using a transgenic mouse that expresses the luciferase reporter gene under the control of the doublecortin promoter. In this model, differentiation of neural progenitor cells lights up in living animals placed under a bioluminescence camera (Couillard-Despres et al., 2008). This readout dimmed within days after these mice were injected with eotaxin. The mice were made in the lab of Ludwig Aigner of Paracelsus Medical University in Salzburg, Austria, a coauthor of this study.
“We think these proteins are bad guys in aging,” Villeda said. Eotaxin has its own literature, though hardly any published work in brain (but see Xia et al., 1998; Choi et al., 2008). At the conference, the Stanford scientists presented no experimental data on MCP-1, which is receiving growing attention in AD research (see e.g., Galimberti et al., 2006; Galimberti et al., 2006). In an interview, Villeda noted that experiments with MCP-1 are ongoing, as well as studies with AD models and repeat parabiosis experiments in other mouse strains.
Villeda emphasized that he believes eotaxin and other such signals enter the brain from the periphery. In fact, the neurogenic niche—a specialized microenvironment in the subventricular zone and the dentate gyrus—appears particularly well positioned to receive molecular cues from the blood. These niches occupy a physical space in which the blood-brain barrier appears to be altered. Several recent studies on the microanatomy of the neurogenic niche have concluded that not only do neural stem cells in the niche make intimate contact with the vascular surface, but they also react to soluble factors released from vascular endothelial cells. Stem cells in vivo were reported to proliferate right next to blood vessels. By comparison, the rest of the brain is more solidly walled off from blood by the classic blood-brain barrier comprising pericytes and astrocytes endfeet, both of which cover the outside of the vascular endothelium (Tavazoie et al., 2008; Shen et al., 2008; Kazanis et al., StemBook).
Besides neurogenesis, what else did the scientists observe about the parabiotic mice? The lab did not conduct a comprehensive examination, but Villeda did notice one curious change. By the end of the blood-sharing period, the old mice, which had gone into it with quite a bit of gray hair, had regrown the dark fur of the C57 black 6 strain used in the experiment. They also had lower mortality than their naturally aging fellow mice and looked a little less—well—old. “There is a rejuvenating ability in young blood onto an aged brain,” said Villeda. Then he quipped, “Maybe Dracula was right: Suck young blood and live forever.”—Gabrielle Strobel.