06 Nov 2015

Numerous studies hint that exercise benefits the brain, but figuring out how has been a hard slog. Confounding factors riddle human results, complicating their interpretation. Enter the lean, mean, mouse machine. According to a report published October 29 in PLOS Biology, exercise prevented age-related breakdown of the brain vasculature in wild-type mice, and staved off the infiltration of inflammatory microglia. Animals lacking ApoE did not enjoy these benefits of “working out,” however. The findings reveal a complex interplay between aging, neurovascular function, and ApoE expression, and illuminate ways in which exercise might tip the balance away from decline. The researchers, led by Gareth Howell at the Jackson Laboratory in Bar Harbor, Maine, have yet to determine how human ApoE alleles, which differ from the rodent isoform, fit into the picture.

“These findings put ApoE and innate immunity at the crossroads of exercise and blood-brain barrier integrity,” said Terrence Town of the University of Southern California in Los Angeles. “It seems that the lion’s share of exercise benefits come from ApoE, at least in mice.” Town and other commentators pointed out that the story could unfold differently in humans because of their specific ApoE isoforms.

While their methods and findings vary greatly, many studies point to the cognitive benefits of exercise and even suggest that regular workouts may stave off Alzheimer’s disease (see July 2014 conference news; Aug 2015 conference news; and AlzRisk analysis). It remains to be seen how working up a sweat benefits cognition, but some evidence suggests that preservation of cerebral blood flow and reduced hypoxia may be involved (see Ainslie et al., 2008; Paillard et al., 2015).

To get a better handle on the subject, first author Ileana Soto and colleagues turned to mice. They first compared gene expression profiles of four-month-old and 21-month-old mice in several regions of the brain. A strong pattern emerged in the cortex: Genes involved in vascular function changed dramatically with age. The shifts were less pronounced in the hippocampus, however, the researchers noted that the vascular density there is lower than in the cortex, potentially masking vascular-related changes in gene expression in that region. In the cortex of aged mice, a threefold increase in deposits of the plasma protein fibrinogen, both in and outside of blood vessels, confirmed that the vasculature was compromised.

Changing of the Guard. In aged mice (right), pericytes (pink) decline while microglia (green) rush in. [Courtesy of Soto et al., PLOS Biology 2015.]

The researchers next zoomed in on the vasculature to further decipher age-related changes. Compared to young mice, aged mice lacked collagen IV and laminin, two key components of the basement membrane that supports the barrier, in the cortex and hippocampus. Immunostaining detected 20 percent fewer pericytes in the cortex, and they covered only half as many of the blood vessels there. Pericytes bolster the blood-brain barrier by preventing transcytosis across endothelial cells that line vessel walls, and they facilitate communication between endothelial cells and astrocytes. Astrocytes in aged mice expressed more glial fibrillary acidic protein (GFAP), a marker of reactivity, than those in younger mice, and expressed lower levels of aquaporin-4 (AQP4), a protein that regulates water transport in the brain and has been linked to clearance of solutes such as amyloid-β (see Aug 2012 news).

Aged mice also had elevated numbers of microglia associated with blood vessels. Electron micrographs of cortical sections revealed that these microglia engulfed apoptotic cell debris, and tended to congregate in regions with fibrin deposits and degenerating pericytes. Aged mice had three times as many microglia expressing the complement protein C1qA, which has emerged as a major player in microglial-dependent pruning of synapses in AD and other diseases (see Nov 2015 conference news). Together, these findings indicate that aging alters key components of the neurovasculature and elevates neuroinflammation around blood vessels.

Freewheeling Mice Fight Age. Mice that voluntarily exercised fended off neurovascular changes as they aged. [Courtesy of Soto et al., PLOS Biology 2015.]

Might exercise alter these age-related vascular changes? To find out, the researchers compared the neurovasculature in 18-month-old sedentary mice to animals of the same age that were given free access to a running wheel at 12 months. At 18 months, the runners performed like middle-aged mice in several measures of mouse daily living, including grip strength, nest construction, and burrowing behavior, while their sedentary counterparts had declined. In the elderly exercised mice, most components of the neurovasaculature—including basement membrane proteins, pericyte coverage, astrocyte reactivity, and microglial infiltration and complement expression—looked very similar to those in four-month-old mice. Running also prevented age-related reduction in the synaptic proteins synaptophysin and PSD-95. However, aged mice that never exercised displayed all the characteristic signs of neurovascular decline. Interestingly, exercise did not protect ApoE knockout mice. Instead, they looked the same as their sedentary brethren. However, because ApoE knockouts had already displayed neurovascular changes by 12 months of age, it is possible that exercise was unable to reverse this damage once it had begun, the researchers noted. Complicating interpretation even more, the initial gene profiling experiments revealed that ApoE expression in the cortex and hippocampus plummeted with age in normal mice. Could exercise reverse that? Further analysis indicated that astrocytes were the primary producers of ApoE in these brain regions, and that exercise prevented their age-related decline in ApoE production. The researchers concluded that loss of ApoE with age may lead to neurovascular changes, and that exercise may rescue this effect by somehow boosting ApoE expression.

The paper’s findings linking cerebrovascular and neuroinflammatory changes with the aging process confirm what many other studies have reported, commented Costantino Iadecola of Weill Cornell Medical College in New York. Cynthia Lemere of Brigham and Women’s Hospital in Boston agreed. She, too, noted a preponderance of findings from her own lab and others pointing to neurovascular decline and the damaging role of the complement cascade with age or during neurodegenerative disease (see Janota et al., 2015; Shi et al., 2015; Stephan et al., 2013). In a joint comment to Alzforum, Axel Montagne and Berislav Zlokovic of the University of Southern California in Los Angeles pointed out that the mouse study meshes with their recent findings linking BBB damage, as well as pericyte degeneration, with age and cognitive decline in people (see Feb 2015 Webinar; and Sagare et al., 2015).

Nga Bien-Ly of Genentech in South San Francisco, who recently reported that the blood-brain barrier remains intact in mouse models of neurodegenerative disease, commented that more quantitative measures of BBB leakage could strengthen the mostly histological observations of the study (see Oct 2015 news).

All commentators agreed that the novel role of ApoE in manifesting the neurovascular benefits of exercise was the most important finding of the paper.

“What we now need to understand is the relationship between astrocytes, ApoE, and complement signaling through microglia,” Lemere said. “They must be involved in some feedback mechanism that ultimately leads to neurovascular dysfunction and cognitive decline, but we don’t know how it all ties together.”

Howell also sees astrocytes, which interact with myriad cells in the brain, as central players. He intends to use an army of transgenic mice to tease out the role of these and other cells in greater detail. He plans to use mice that express different isoforms of human ApoE to determine whether they modulate exercise differently. He also intends to use mice in which ApoE expression is switched off just as the animals embark on their midlife exercise routines to distinguish between pre- and post-exercise effects of the protein. Further studies in mice lacking complement proteins, or in animals in which resident microglia can be distinguished from peripheral monocytes, could further elucidate the role of microglia in this process, Howell told Alzforum.

How does any of this relate to AD? Howell is interested in that, but said that a deeper understanding of the changes that accompany aging—the primary risk factor for AD—is what is most needed in the field. Interestingly, a previous study reported that exercise reduced amyloid deposition people who carry an ApoE4 allele, but had no effect in non-carriers (although non-carriers had lower amyloid burden to begin with) (see Head et al., 2012).

In their simplest interpretation, Lemere said the findings should at least motivate people to exercise. She added, “I plan to go on a brisk walk immediately following this call.”—Jessica Shugart