Zebrafish use surprising strategy to regrow spinal cord

Zebrafish are members of a rarefied group of vertebrates capable of fully healing a severed spinal cord. A clear understanding of how this regeneration takes place could provide clues toward strategies for healing spinal cord injuries in people. Such injuries can be devastating, causing permanent loss of sensation and movement.

A new study from Washington University School of Medicine in St. Louis maps out a detailed atlas of all the cells involved -- and how they work together -- in regenerating the zebrafish spinal cord. In an unexpected finding, the researchers showed that survival and adaptability of the severed neurons themselves is required for full spinal cord regeneration. Surprisingly, the study showed that stem cells capable of forming new neurons -- and typically thought of as central to regeneration -- play a complementary role but don't lead the process.

The study is published Thursday, Aug. 15, in the journal Nature Communications.

Unlike humans' and other mammals' spinal cord injuries, in which damaged neurons always die, the damaged neurons of zebrafish dramatically alter their cellular functions in response to injury, first to survive and then to take on new and central roles in orchestrating the precise events that govern healing, the researchers found. Scientists knew that zebrafish neurons survive spinal cord injury, and this new study reveals how they do it.

"We found that most, if not all, aspects of neural repair that we're trying to achieve in people occur naturally in zebrafish," said senior author Mayssa Mokalled, PhD, an associate professor of developmental biology. "The surprising observation we made is that there are strong neuronal protection and repair mechanisms happening right after injury. We think these protective mechanisms allow neurons to survive the injury and then adopt a kind of spontaneous plasticity -- or flexibility in their functions -- that gives the fish time to regenerate new neurons to achieve full recovery. Our study has identified genetic targets that will help us promote this type of plasticity in the cells of people and other mammals."

By mapping out the evolving roles of various cell types involved in regeneration, Mokalled and her colleagues found that the flexibility of the surviving injured neurons and their capacity to immediately reprogram after injury lead the chain of events that are required for spinal cord regeneration. If these injury-surviving neurons are disabled, zebrafish do not regain their normal swim capacity, even though regenerative stem cells remain present.

When the long wiring of the spinal cord is crushed or severed in people and other mammals, it sets off a chain of toxicity events that kills the neurons and makes the spinal cord environment hostile against repair mechanisms. This neuronal toxicity could provide some explanation for the failure of attempts to harness stem cells to treat spinal cord injuries in people. Rather than focus on regeneration with stem cells, the new study suggests that any successful method to heal spinal cord injuries in people must start with saving the injured neurons from death.

"Neurons by themselves, without connections to other cells, do not survive," Mokalled said. "In zebrafish, we think severed neurons can overcome the stress of injury because their flexibility helps them establish new local connections immediately after injury. Our research suggests this is a temporary mechanism that buys time, protecting neurons from death and allowing the system to preserve neuronal circuitry while building and regenerating the main spinal cord."

There is some evidence that this capacity is present but dormant in mammalian neurons, so this may be a route to new therapies, according to the researchers.

"We are hopeful that identifying the genes that orchestrate this protective process in zebrafish -- versions of which also are present in the human genome -- will help us find ways to protect neurons in people from the waves of cell death that we see following spinal cord injuries," she said.

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Can hearing loss be reversed? Research reveals clues that could regrow the cells that help us hear

Taking a bite of an apple is considered a healthy choice. But have you ever thought about putting in earplugs before your favorite band takes the stage?

Just like your future body will thank you for the apple, your future ears (specifically your cochlear hair cells) will thank you for protecting them. The most common cause of hearing loss is progressive because these hair cells -- the primary cells to detect sound waves -- cannot regenerate if damaged or lost. People who have repeated exposure to loud noises, like military personnel, construction workers, and musicians, are most at risk for this type of hearing loss. But, it can happen to anyone over time (even concert goers).

On the other hand, birds and fish can regenerate these hair cells, and now researchers at the Del Monte Institute for Neuroscience are getting closer to identifying the mechanisms that may promote this type of regeneration in mammals, as explained in research recently published in Frontiers in Cellular Neuroscience.

"We know from our previous work that expression of an active growth gene, called ERBB2, was able to activate the growth of new hair cells (in mammals), but we didn't fully understand why," said Patricia White, PhD, professor of Neuroscience and Otolaryngology at the University of Rochester Medical Center. The 2018 study led by Jingyuan Zhang, PhD, a postdoctoral fellow in the White lab at the time, found that activating the growth gene ERBB2 pathway triggered a cascading series of cellular events by which cochlear support cells began to multiply and activate other neighboring stem cells to become new sensory hair cells.

"This new study tells us how that activation is happening -- a significant advance toward the ultimate goal of generating new cochlear hair cells in mammals," said White.

Using single-cell RNA sequencing in mice, researchers compared cells with an overactive growth gene (ERBB2 signaling) with similar cells that lacked such signaling. They found the growth gene -- ERBB2 -- promoted stem cell-like development by initiating the expression of multiple proteins -- including SPP1, a protein that signals through the CD44 receptor. The CD44 receptor is known to be present in cochlear-supporting cells. This increase in cellular response promoted mitosis in the supporting cells, a key event for regeneration.

"When we checked this process in adult mice, we were able to show that ERBB2 expression drove the protein expression of SPP1 that is necessary to activate CD44 and grow new hair cells," said Dorota Piekna-Przybylska, PhD, a staff scientist in the White Lab and first author of the study. "This discovery has made it clear that regeneration is not only restricted to the early stages of development. We believe we can use these findings to drive regeneration in adults."

"We plan to further investigation of this phenomenon from a mechanistic perspective to determine whether it can improve auditory function after damage in mammals. That is the ultimate goal," said White.

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Ancient disease has potential to regenerate livers

Leprosy is one of the world's oldest and most persistent diseases but the bacteria that cause it may also have the surprising ability to grow and regenerate a vital organ.

Scientists have discovered that parasites associated with leprosy can reprogramme cells to increase the size of a liver in adult animals without causing damage, scarring or tumors.

The findings suggest the possibility of adapting this natural process to renew ageing livers and increase healthspan -- the length of time living disease-free -- in humans.

Experts say it could also help regrow damaged livers, thereby reducing the need for transplantation, which is currently the only curative option for people with end-stage scarred livers.

Previous studies promoted the regrowth of mouse livers by generating stem cells and progenitor cells -- the step after a stem cell that can become any type of cell for a specific organ -- via an invasive technique that often resulted in scarring and tumour growth.

To overcome these harmful side-effects, Edinburgh researchers built on their previous discovery of the partial cellular reprogramming ability of the leprosy-causing bacteria, Mycobacterium leprae.

Working with the US Department of Health and Human Services in Baton Rouge, Louisiana, the team infected 57 armadillos -- a natural host of leprosy bacteria -- with the parasite and compared their livers with those of uninfected armadillos and those that were found to be resistant to infection.

They found that the infected animals developed enlarged -- yet healthy and unharmed -- livers with the same vital components, such as blood vessels, bile ducts and functional units known as lobules, as the uninfected and resistant armadillos.

The team believe the bacteria 'hijacked' the inherent regenerative ability of the liver to increase the organ's size and, therefore, to provide it with more cells within which to increase.

They also discovered several indicators that the main kinds of liver cells -- known as hepatocytes -- had reached a "rejuvenated" state in the infected armadilllos.

Livers of the infected armadillos also contained gene expression patterns -- the blueprint for building a cell -- similar to those in younger animals and human fetal livers.

Genes related to metabolism, growth and cell proliferation were activated and those linked with aging were downregulated, or suppressed.

Scientists think this is because the bacteria reprogramed the liver cells, returning them to the earlier stage of progenitor cells, which in turn became new hepatocytes and grow new liver tissues.

The team are hopeful that the discovery has the potential to help develop interventions for aging and damaged livers in humans. Liver diseases currently result in two million deaths a year worldwide.

The findings have been published in the journal Cell Reports Medicine. This work has been funded by the UK's Medical Research Council and the US National Institutes of Health and National Institute of Allergy and Infectious Diseases.

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From wound healing to regeneration

The phenomenon of regeneration was discovered over 200 years ago in the freshwater polyp Hydra. Until now, however, it was largely unclear how the orderly regeneration of lost tissues or organs is activated after injury. In its investigations of Hydra, an interdisciplinary research team at Heidelberg University was able to show how wound healing signals released upon injury are converted into specific signals of pattern formation and cell differentiation. Essential components are the mitogen-activated protein kinases (MAPK) and the Wnt signalling pathway -- molecular mechanisms that have remained relatively unchanged throughout evolution.

The ability to regenerate varies widely in animals. Most mammals and vertebrates have only limited regeneration capacity, while basal and simple animals that emerged early in evolution, like cnidarians and planarians, can regenerate their whole body. In all cases, the process of regeneration begins with wound healing. The cells at the site of injury proliferate and form an undifferentiated mass -- a blastema -- from which the missing structures are re-patterned. This activates genetic processes that also control embryonic development. To determine the molecular mechanisms involved, the research team led by Prof. Dr Thomas W. Holstein studied the freshwater polyp Hydra to understand the basic features of this activation of regeneration.

The core of their investigations is the doctoral thesis of Anja Tursch. She repeated the key experiment of Geneva naturalist Abraham Trembley (1710 to 1784) which led him to discover the regeneration phenomenon. The Hydra polyp is bisected, prompting the upper half to regenerate a new "head" and the lower half a new "foot" -- hence totally different body parts can grow from the exact same tissue at the cut surface in the middle. Building on their previous work on Hydra regeneration, the researchers at the Centre for Organismal Studies (COS) of Heidelberg University have now shown how this is possible.

Regardless of where it occurs, any damage triggers nonspecific signals for an injury response, i.e. wound healing, via calcium ions and the production of reactive oxygen species. The signals are transmitted intracellularly by three mitogen-activated protein kinases -- p38, JNKs, and ERK. Activation of these three molecules is required for both head and foot regeneration. Wnt signalling pathways are then activated that are important during embryonic development for the formation of rudimentary organs and the body axis. The generic signals of wound healing are thus transferred into position-specific signals of patterning and cell differentiation for regeneration.

"Our experiments show that the Wnt signalling pathway is a main component of the initially general injury response and, depending on signal strength, directs the tissue toward head or foot development," explains Prof. Holstein. This is why, in the case of MAPK inhibition, the otherwise absent regeneration can be induced by artificially generated, recombinant Wnt proteins. "It was also surprising that in middle body parts that had both head and foot removed, heads can be induced at both ends in this way," adds Dr Suat Özbek, a member of Prof. Holstein's "Molecular Evolution and Genomics" research group at the COS.

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New research challenges long-held beliefs about limb regeneration

Ken Muneoka is no stranger to disrupting the field of regeneration; for example, in a 2019 ground-breaking publication in Nature, the Texas A&M University College of Veterinary Medicine & Biomedical Sciences (CVMBS) professor proved for the first time that joint regeneration in mammals was possible.

Now, his team is again challenging other centuries-old beliefs about the fundamental science of the field, this time related to how mammals might regenerate damaged parts of the body.

In humans, the natural ability to regenerate is limited to tissues like the epidermis, the outermost layer of skin, and some organs, such as the liver.

Other species, most notably salamanders, have the ability to regenerate complex structures such as bones, joints, and even entire limbs. As a result, scientists have been studying these species for more than 200 years to try to understand the mechanisms behind limb regeneration in the hopes of someday translating those mechanisms to induce more extensive regeneration in humans.

That research has led to a common belief that the single biggest key for limb regeneration is the presence of nerves.

While that may be true for salamanders and other species, it isn't the case in mammals, according to two of Muneoka's recently published studies. The first study, published last year in the Journal of Bone and Mineral Research, established that mechanical loading (the ability to apply force to or with an affected area) is a requirement for mammals. The second, published earlier this year in Developmental Biology, established that the absence of nerves does not inhibit regeneration.

Together, these findings present a sizeable shift in the thinking of how regeneration could work in human medicine.

"What these two studies show counteracts the two-century-old dogma that you need nerves to regenerate," Muneoka said. "What replaces it in mammals is that you need mechanical loading, not nerves."

Importance Of Mechanical Load

Scientists have long believed that two things must be present in an affected area in order to induce regeneration in mammals. The first is growth factors, which are molecules that can stimulate cells to regrow and reconstruct parts of the body.

In natural regeneration, these growth factors, which vary from species to species and by area being regenerated, are produced by the body. For human-induced regeneration, these growth factors must be introduced to the area.

The second factor believed to be necessary was nerves. This belief was predicated by many previous human-induced mammal regeneration studies on areas, usually digit tips, without nerves, in which the whole limbs were also no longer usable.

Those studies would have the predicted outcome -- when growth factors were introduced regeneration did not take place-leading to the conclusion that, like in other species, nerves were a requirement for regeneration.

But the mechanical load aspect was ignored.

In their studies, Muneoka and colleagues decided to take a step back and ask the question, "is it really the nerves, or is lack of mechanical load part of the equation as well?"

Connor Dolan, a former graduate student in Muneoka's lab and first author on both new studies (who now works at the Walter Reed National Military Medical Center), came up with a way to test the denervation requirement in mammals that was inspired by astronauts.

The technique, called hindlimb suspension, has been used by NASA and other scientists for decades to test how mammals react to zero gravity environments. A similar process is used during medical procedures on legs of large animals to prevent the animals from putting weight on the affected limbs.

"Dolan found that when the limbs were suspended, even though they still had lots of nerves and could move around, they couldn't actually put pressure on their limbs so the digit tips wouldn't regenerate," Muneoka said. "It just completely inhibited regeneration."

As soon as the mechanical load returns, however, regeneration is rescued.

"Absolutely nothing happens during the suspension," Muneoka said. "But once the load returns, there will be a couple weeks of delay, but then they'll begin to regenerate."

That first step proved that even though nerves might be required, the mechanical loading was a critical component to regeneration.

Taking the research a step further, Dolan's second publication showed that nerves weren't required by demonstrating that if a mouse has no nerves in one of its digits but does in the others -- so that it's still exerting force on the denervated digit -- that digit will still regenerate.

"He found that they regenerate a little bit slower, but they regenerated perfectly normally," Muneoka said.

Ramifications Of The Research

Muneoka is quick to point out that their studies aren't saying that previous research is wrong, just that it doesn't directly apply to humans.

"There have been a number of studies in salamanders that prove that when you remove the nerves, they do not regenerate," Muneoka said. "Researchers have also been able to put growth factors they know are being produced by nerves into the cells and rescue regeneration.

"So, salamanders probably do need nerves to regenerate," he said. "But if we're going to regenerate limbs in humans, it's going to be a lot more like what happens in mice."

Since first beginning to look at regeneration more than 20 years ago, a number of Muneoka's ideas have pushed back against the generally accepted theories about regeneration. He said that getting these two papers published took almost three years because they originally tried to submit them together.

"Many scientists don't embrace this idea," he said. "A lot of people's careers are really dependent on their studies of nerves and how they affect regeneration. For a study to come out and say that for humans it's unlikely you'll need the nerves, the whole biomedical application of what people are doing in salamanders and fish kind of goes out the window."

Looking Down The Road

Nerves not being required for regeneration in mammals may seem like an academic point. After all, what would be the point of regenerating a limb if the person couldn't feel it or control it because it had no nerves. In that sense, nerves are still going to be an important part of the puzzle.

From Muneoka's perspective, the shift is that instead of thinking of nerves as a requirement for regeneration, nerves are a part of what needs to be regenerated.

Larry Suva, head of the CVMBS' Department of Veterinary Physiology & Pharmacology (VTPP), says the issue is that nobody was even thinking about the load aspect previously.

"Think of a blast injury where a soldier is left with a stump," Suva said. "No one, until this paper came out, was even thinking about a requirement from mechanical influences. You had people see that a denervated animal doesn't regenerate and they're thinking it's because the nerve was cut, but nobody was studying the mechanical load aspect."

As Suva puts it, science is full of people looking where the light is best.

"I work on bones, so when I see a problem, I look at the bone problem," he said. "People who work on nerves, all they look at are nerves. So it's very rare that someone like Dr. Muneoka will take a step back and take a more holistic view.

"That's what he brought to this idea, to this 200-year-old data," Suva said. "We now have to look at regeneration through a different lens because now we know the mechanical influences are extremely important."

One of the results of research focusing on nerves is that scientists have been able to recreate the growth factors that nerves produce, which has allowed researchers to start regeneration in salamanders, even if the nerves aren't present. Suva said that with these new findings, scientists will now know they have to do the same with the mechanical load aspect if they want to start regeneration in mammals.

"Scientists already have been able to trick the body into thinking nerves are still present," he said. "But now they know they'll also have to trick it into thinking there's a mechanical load, something that has not been done before."

Because cells react differently under mechanical load, somehow, that load is being translated biochemically inside the cell.

"There's a small number of labs looking at the biochemical basis for what mechanical load does to a cell," Muneoka said. "If we could understand that biochemical signal, then perhaps the physical force of mechanical load can be replaced by some sort of cocktail of molecules that will create the same signals in the cells."

The end of the road toward full human regeneration may still be a long way in the future, but Suva says that this kind of fundamental shift in thinking is a major marker on that road.

"Regeneration of a human limb may still be science fiction, but we know some facts about it, and now we know you have to have that mechanical load along with the growth factors," he said. "That changes how future scientists and engineers are going to solve this problem.

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Scientists regrow frog's lost leg

For millions of patients who have lost limbs for reasons ranging from diabetes to trauma, the possibility of regaining function through natural regeneration remains out of reach. Regrowth of legs and arms remains the province of salamanders and superheroes.

But in a study published in the journal Science Advances, scientists at Tufts University and Harvard University's Wyss Institute have brought us a step closer to the goal of regenerative medicine.

On adult frogs, which are naturally unable to regenerate limbs, the researchers were able to trigger regrowth of a lost leg using a five-drug cocktail applied in a silicone wearable bioreactor dome that seals in the elixir over the stump for just 24 hours. That brief treatment sets in motion an 18-month period of regrowth that restores a functional leg.

Many creatures have the capability of full regeneration of at least some limbs, including salamanders, starfish, crabs, and lizards. Flatworms can even be cut up into pieces, with each piece reconstructing an entire organism. Humans are capable of closing wounds with new tissue growth, and our livers have a remarkable, almost flatworm-like capability of regenerating to full size after a 50% loss.

But loss of a large and structurally complex limb -- an arm or leg -- cannot be restored by any natural process of regeneration in humans or mammals. In fact, we tend to cover major injuries with an amorphous mass of scar tissue, protecting it from further blood loss and infection and preventing further growth.

Kickstarting Regeneration

The Tufts researchers triggered the regenerative process in African clawed frogs by enclosing the wound in a silicone cap, which they call a BioDome, containing a silk protein gel loaded with the five-drug cocktail.

Each drug fulfilled a different purpose, including tamping down inflammation, inhibiting the production of collagen which would lead to scarring, and encouraging the new growth of nerve fibers, blood vessels, and muscle. The combination and the bioreactor provided a local environment and signals that tipped the scales away from the natural tendency to close off the stump, and toward the regenerative process.

The researchers observed dramatic growth of tissue in many of the treated frogs, re-creating an almost fully functional leg. The new limbs had bone structure extended with features similar to a natural limb's bone structure, a richer complement of internal tissues (including neurons), and several "toes" grew from the end of the limb, although without the support of underlying bone.

The regrown limb moved and responded to stimuli such as a touch from a stiff fiber, and the frogs were able to make use of it for swimming through water, moving much like a normal frog would.

"It's exciting to see that the drugs we selected were helping to create an almost complete limb," said Nirosha Murugan, research affiliate at the Allen Discovery Center at Tufts and first author of the paper. "The fact that it required only a brief exposure to the drugs to set in motion a months-long regeneration process suggests that frogs and perhaps other animals may have dormant regenerative capabilities that can be triggered into action."

The researchers explored the mechanisms by which the brief intervention could lead to long-term growth. Within the first few days after treatment, they detected the activation of known molecular pathways that are normally used in a developing embryo to help the body take shape.

Activation of these pathways could allow the burden of growth and organization of tissue to be handled by the limb itself, similar to how it occurs in an embryo, rather than require ongoing therapeutic intervention over the many months it takes to grow the limb.

How the BioDome Works

Animals naturally capable of regeneration live mostly in an aquatic environment. The first stage of growth after loss of a limb is the formation of a mass of stem cells at the end of the stump called a blastema, which is used to gradually reconstruct the lost body part. The wound is rapidly covered by skin cells within the first 24 hours after the injury, protecting the reconstructing tissue underneath.

"Mammals and other regenerating animals will usually have their injuries exposed to air or making contact with the ground, and they can take days to weeks to close up with scar tissue," said David Kaplan, Stern Family Professor of Engineering at Tufts and co-author of the study. "Using the BioDome cap in the first 24 hours helps mimic an amniotic-like environment which, along with the right drugs, allows the rebuilding process to proceed without the interference of scar tissue."

Next Steps in Frogs and Mammals

Previous work by the Tufts team showed a significant degree of limb growth triggered by a single drug, progesterone, with the BioDome. However, the resulting limb grew as a spike and was far from the more normally shaped, functional limb achieved in the current study.

The five-drug cocktail represents a significant milestone toward the restoration of fully functional frog limbs and suggests further exploration of drug and growth factor combinations could lead to regrown limbs that are even more functionally complete, with normal digits, webbing, and more detailed skeletal and muscular features.

"We'll be testing how this treatment could apply to mammals next," said corresponding author Michael Levin, Vannevar Bush Professor of Biology in the School of Arts & Sciences, director of the Allen Discovery Center at Tufts, and associate faculty member of the Wyss Institute.

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Tiny salamander's huge genome may harbor the secrets of regeneration

Axolotl

The type of salamander called axolotl, with its frilly gills and widely spaced eyes, looks like an alien and has other-worldly powers of regeneration. Lose a limb, part of the heart or even a large portion of its brain? No problem: They grow back.

"It regenerates almost anything after almost any injury that doesn't kill it," said Parker Flowers, postdoctoral associate in the lab of Craig Crews, the John C. Malone Professor of Molecular, Cellular, and Developmental Biology and professor of chemistry and pharmacology.

If scientists can find the genetic basis for the axolotl's ability to regenerate, they might be able to find ways to restore damaged tissue in humans. But they have been thwarted in the attempt by another peculiarity of the axolotl -- it has the largest genome of any animal yet sequenced, 10 times larger than that of humans.

Now Flowers and colleagues have found an ingenious way to circumvent the animal's complex genome to identify at least two genes involved in regeneration, they report Jan. 28 in the journal eLife.

The advent of new sequencing technologies and gene-editing technology has allowed researchers to craft a list of hundreds of gene candidates that could responsible for regeneration of limbs. However, the huge size of the axolotl genome populated by vast areas of repeated stretches of DNA has made it difficult to investigate the function of those genes.

Lucas Sanor, a former graduate student in the lab, and fellow co-first author Flowers used gene editing techniques in a multi-step process to essentially create markers that could track 25 genes suspected of being involved in limb regeneration. The method allowed them to identify two genes in the blastema -- a mass of dividing cells that form at the site of a severed limb -- that were also responsible for partial regeneration of the axolotl tail.

Flowers stressed that many more such genes probably exist. Since humans possess similar genes, the researchers say, scientists may one day discover how to activate them to help speed wound repair or regenerate tissue.

From Science Daily