On a rain-tossed night in 1816, while visiting the Swiss villa of her friend the poet Lord Byron, 19-year-old Mary Shelley had a dream that would inspire one of the best-known stories in literature. “I saw the pale student of unhallowed arts kneeling beside … a hideous phantasm of a man,” she wrote in the preface to Frankenstein. Though she does not say in her novel how exactly the creature—a disquieting patchwork of human body parts—is brought to life, she would later write that her book, and the vision on which it was based, were inspired by the concept of galvanism: that the wounded could be healed and the dead possibly returned to life by the application of currents of electricity.
A belief in the regenerative powers of electricity—investigated by such luminaries as Benjamin Franklin, after whom, some say, Frankenstein was named—fell into disrepute for much of the 20th century. The concept was revived in the 1980s when a band of researchers showed they could repair severed spinal cords in guinea pigs and rats by running currents across the injured tissue. But the findings and, indeed, the whole field of bioelectricity were pushed to the side by the onrush of molecular biology.
In a paper that could help bring the study of bioelectricity into the mainstream of 21st century science, Forsyth Institute researchers have identified a protein that serves as a natural source of regenerative electricity. By manipulating the protein, an ion transporter, they were able to induce frog tadpoles to regrow tails at a stage of development when such regrowth is typically not possible.
The researchers say that the findings, which appear in the Feb. 28 online Development, could lead to a whole new way of repairing and regrowing injured spinal cords and other damaged tissue.
Forging a Link
What had been missing from studies until now is an understanding of how electricity—the flow of charged particles—works at a molecular level to bring about regeneration. Can a change in the way ions are distributed in concentration gradients across cells and tissues actually turn genes on and off and lead to cell proliferation and the other changes required for regeneration? Also lacking was an understanding of what molecular agents might be directing the formation of such gradients.
Dany Adams, Michael Levin, and Alessio Masi explored these questions in a familiar model, Xenopus, the African clawed frog. Xenopus tadpoles, like those of many types of frog, can regrow severed tails except during a stage known as the refractory period. Masi, then a postdoctoral fellow, and Adams, an HSDM research associate in developmental biology, blocked a variety of ion transporters, proteins that ferry ions across the cell membrane. Interrupting the activity of one of these, the ion pump V-ATPase, left the tadpoles unable to regenerate tails, even during the normally permissive period. The researchers tried rescuing the animals, giving them a different hydrogen ion pump. The tails regrew.
In a subsequent experiment, Adams was able to get xenopus tails to regrow even during the refractory period. The regenerated appendages exhibited the same intricate patterning found in normal tadpole tails.
“This is hugely important—the tail is a very complex structure,” said Levin, HSDM assistant professor of developmental biology and director of the Forsyth Center for Regenerative and Developmental Biology. “It has muscle, spinal cord, blood vessels. We’ve provided one bit of information: hydrogen flow ‘on.’ From there you get the whole tail. It’s like what they call a master regulator in other contexts. It’s something that kicks off a cascade of things that the embryo already knows how to do. And it stops when it is done.”
Mimicking this natural body intelligence, Levin said, could lead to a revival of the centuries-old dream of galvanism—using electricity to regenerate damaged or torn tissue. It is a dream that might be closer to reality than many realize. Purdue researcher Richard Borgens, who, in the 1980s, used electric currents to regenerate tissue in rodents, has since conducted studies in other animals. Over the past decade, he and his colleagues have implanted batteries into the backs of dogs with accident-related spinal-cord injuries, with promising results. The approach is currently in human clinical trials.
“My image of why our study is so exciting is that the closer you can get to the way the organism does it itself, the more likely you are able to both start something, but also have the organism control it properly and turn it off at the right time,” said Adams. “Putting a battery across a severed cord, that is great, but there are all kinds of issues. How strong a battery? When do you take the battery off? What is actually happening? And is the body going to be able to regulate it properly? By doing it with gene therapy and going to the molecular light switch, the body may very well be able to regulate it. And it takes away a huge and possibly very complicated series of side effects.”
Currents of Regeneration
Levin and his colleagues were well-poised to identify the electrical switch controlling Xenopus tail regeneration. In 2002, they showed that the development of left–right asymmetry in xenopus embryos is determined by a gradient-establishing hydrogen ion pump (see Focus, Oct. 11, 2002). They turned to another distinctive feature of Xenopus development, tail regeneration, and asked, might ion transporters be playing a role here too? Using reagents developed in their earlier experiments, they ran a screen of ion transporter blockers that suggested V-ATPase, along with two other ion transporters, might be involved.
To test their result, they inhibited V-ATPase activity using a dominant-negative construct. The xenopus tadpoles were unable to regenerate their tails though they were able to heal wounds and, in other respects, continued to develop normally. Adams and colleagues tried rescuing the animals by introducing mRNA for a hydrogen pump derived from yeast. The animals regrew their tails.
To determine where V-ATPase was setting up its growth-restoring flow of ions, Adams exposed amputated xenopus tails to voltage-sensitive dyes. She found that ions were being pumped right at the very edge of the wound, by the freshly injured cells, and the cells were doing so only six hours after amputation. She made other intriguing observations. Normally, the Xenopus tail is polarized. The tail becomes depolarized upon amputation, but is repolarized after a day. Adams found that when V-ATPase was blocked, repolarization did not occur.
Perhaps the most exciting findings came when she expressed the yeast pump in tadpoles during the refractory period. The tadpoles were able to regenerate their tails, and the regeneration was correlated with a repolarization. “That was really the kicker,” said Levin.
In further experiments, the researchers found that V-ATPase activity was crucial for inducing three activities required for regeneration: increased cell proliferation, the turning on of critical downstream genes, and the orderly setting up of axon pathways (see figure).
Tracing the downstream effects of V-ATPase and other ion transporters will be an important task. “The idea ultimately in all this is that by understanding what you want in the bioelectrical properties of a regenerating tissue, you could make that happen by putting in the appropriate ion transporters,” said Levin. The benefits may not be limited to patients with spinal cord and other tissue injuries. Getting cells to proliferate—or stop proliferating—is a goal of many
branches of medicine.
“The field as a whole has applications to cancer biology because tumors have different voltage properties than normal cells,” he said. “Almost any tissue where you can modulate cell behavior through these kinds of mechanisms is going to be potentially amenable to some kind of shift in proliferative and differentiation potential.”