In the healthy central nervous system, blood flows where neurons need it most. Now, researchers led by Adriana Di Polo at the University of Montreal help explain why. In the August 12 Nature, they describe a new mechanism for fine control of neurovascular coupling in the retinae of living mice. Delicate tubes connecting pericytes on adjoining capillaries coordinate regional blood flow, they find.
- Tunneling nanotubes connect pericytes on different capillaries in mouse retinae.
- In response to light, one pericyte constricts its capillary, the other widens.
- The coordinated action depends on calcium waves traveling through the nanotube.
These thread-like structures looked like tunneling nanotubes. They extended from one cell, formed a gap junction at the distal end with another, and were packed with cytoplasm and organelles. Calcium waves propagated in either direction through the tubes, suggesting the linked pericytes were communicating. In response to neuronal activation, nearby pericytes relaxed to let blood vessels dilate, while their distal, connected partners contracted, constricting blood flow where it was not needed. This coordinated but opposing action may help channel blood in the retina, where supply is limited, to the most active neurons, the authors believe. “This is the first time a physiological role for any tunneling nanotube has been demonstrated in vivo,” Di Polo told Alzforum.
Chiara Zurzolo at the Pasteur Institute in Paris called the findings significant, and the morphological and functional evidence for the structures impressive. “By using state-of-the-art imaging approaches, Di Polo and collaborators provide the first evidence for the existence of closed tunneling-nanotube-like protrusions in the retina of living mice,” Zurzolo wrote to Alzforum. She also wrote a News & Views for the paper.
Tunneling nanotubes were first described in cell culture by Hans-Hermann Gerdes and colleagues at the University of Heidelberg, Germany. These slender ducts, around 200 nm in diameter, transferred organelles between cells (Rustom et al., 2004; Gerdes et al., 2007). Other studies reported that nanotubes coordinate electrical signaling among cultured cells (Wang et al., 2010). However, researchers have debated whether the structures exist in vivo, and what purpose they serve.
Separately, the literature as far back as 1838 contains reports of fine “vascular strands” linking capillaries in fixed retinal tissue, Di Polo said. More recent studies of fixed mouse retinae note the existence of thin “bridging” filaments connecting pericytes on neighboring capillaries (e.g., Mendes-Jorge et al., 2012). But no one had characterized these filaments in living tissue.
Joint first authors Luis Alarcon-Martinez and Deborah Villafranca-Baughman were studying mice whose pericytes are fluorescently labeled when they noticed the fine strands linking these cells in the retina. The tubes were about 500 nm in diameter, surrounded by basement membrane, and linked the cell body of one pericyte, the proximal one, with the process of a distal pericyte on another capillary (see image above). A pericyte extends processes far along its capillary, and that is where the distal end of the nanotube attached, not anywhere near the cell body. About two-thirds of the nanotubes were shorter than 30 microns. About a quarter of retinal pericytes had these structures.
Electron microscopy of fixed retinas revealed that these tubes were stuffed with material, including mitochondria, vesicles, and endoplasmic reticulum. Structurally, they were supported by an F-actin cytoskeleton and contained a contractile protein called α-smooth muscle actin. The connection with the distal pericyte appeared to be a gap junction, a place where two cell membranes butt up against each other, allowing ions and small molecules to pass between them through narrow channels. Supporting this, the connection contained the gap-junction protein connexin 43, and only small molecules were able to diffuse through; organelles and large molecules were not. Adding a gap-junction blocker prevented this exchange. Mitochondria clustered at the gap junction, hinting at a demand for energy there.
Based on these properties, the authors named the structures interpericyte tunneling nanotubes (IP-TNTs). Di Polo noted that they are distinct from the “bridging pericytes” sometimes seen in the retina when a small blood vessel regresses during vascular remodeling. Those leftover connections are thicker, typically several microns in diameter, and are either empty sleeves of basement membrane or are filled with debris and cell fragments.
To find out what tunneling nanotubes do in the eye, Alarcon-Martinez and colleagues used a two-photon laser scanning microscopy technique they had previously developed to study retinae in living mice. The researchers anesthetized the mice, rotated an eyeball and pulled back a flap of sclera to expose the retina, then placed a small coverslip over it. In mice expressing a fluorescent calcium indicator in their pericytes, the authors saw calcium waves traveling in both directions along IP-TNTs. When the researchers stimulated the retina with light, triggering neuronal activity, one of the linked capillaries constricted a few seconds later, while the other dilated. Notably, calcium levels spiked in pericytes on constricting capillaries, and dropped in those on dilating vessels.
“The observation that one vessel constricts and the other dilates was the most unexpected finding in this study,” Di Polo told Alzforum. She noted that reviewers were skeptical at first, because neuronal activity is believed to increase blood flow, not decrease it. Others may have missed this phenomenon because the changes in blood flow are minute, meaning studies in ex vivo tissue would lack the sensitivity to detect them, Di Polo noted. “When you see it in vivo in thousands of pericyte pairs, it’s very convincing and very strong,” she told Alzforum. In the editorial, Zurzolo said it would be interesting to explore whether IP-TNTs mediate neurovascular coupling over larger brain areas.
Costantino Iadecola at Weill Cornell Medical College in New York told Alzforum that if this same mechanism operates in brain, it could help solve the longstanding mystery of how blood is shunted to only the region that needs it after neuronal activation. “This is fantastic in terms of introducing a new concept to explain how this highly interconnected capillary network could be regulated,” he wrote (full comment below).
Is the calcium wave propagation essential for this regulation? The authors disrupted IP-TNTs in living mice with a tightly focused laser on low power, which did not harm surrounding cells. Afterward, the previously linked pericytes no longer synchronized their constriction and dilation, and blood flow no longer changed after light stimulation. Other health conditions had a similar deleterious effect on nanotubes. For example, when the researchers caused transient ischemia in the retina, calcium levels in pericytes shot up, capillaries constricted, and many nanotubes ruptured. Even after blood flow was restored, these damaged areas were no longer able to alter blood flow in response to light.
Importantly, blocking calcium influx into pericytes just before the ischemia prevented damage to IP-TNTs, and they responded normally to light after reperfusion. “Calcium homeostasis is critical to preserving IP-TNT structure and function,” the authors concluded.
These pericyte tendrils are not confined to the retina. The authors found IP-TNTs in mouse visual cortex and other brain regions as well. In ongoing research, they are looking for IP-TNTs in retinal and brain tissue from monkeys and humans.
Martin Lauritzen at the University of Copenhagen, Denmark, noted that the field of brain pericyte physiology is still developing. “Alarcon-Martinez and colleagues have now identified IP-TNT as a potential mechanism that we will need to take into account in further studies of cerebrovascular function and neurovascular coupling in health and disease,” he wrote to Alzforum (see full comment below).
Could IP-TNTs play a role in neurodegenerative disease? To test this, Di Polo and colleagues are infusing Aβ into living mouse retinae to see what effect it might have on nanotubes and pericyte function. Pathogenic proteins such as misfolded prion and α-synuclein have been shown to travel through tunneling nanotubes in other cell types, hinting the structures may help spread pathology (Gousset et al., 2009; Dec 2016 conference news; Dieriks et al., 2017). Aβ deposits have been found in the retinae of AD patients (Jul 2014 conference news; Sep 2017 news; Apr 2019 conference news).—Madolyn Bowman Rogers
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