How Neurons Get Wired

Two different versions of the same signaling protein tell a nerve cell which end is which, UA researchers have discovered. The findings could help improve therapies for spinal injuries and neurodegenerative diseases.

University of Arizona scientists have discovered an unknown mechanism that establishes polarity in developing nerve cells. Understanding how nerve cells make connections is an important step in developing cures for nerve damage resulting from spinal cord injuries or neurodegenerative diseases such as Alzheimer’s.

In a study published on Aug. 12 in the journal Proceedings of the National Academy of Sciences, UA doctoral student Sara Parker and her adviser, assistant professor of cellular and molecular medicine Sourav Ghosh, report that the decision which will be the “plus” and the “minus” end in a newborn nerve cell is made by a long and a short version of the same signaling molecule.

Nerve cells – or neurons – differ from many other cells by their highly asymmetric shape: Vaguely resembling a tree, a neuron has one long, trunk-like extension ending in a tuft of root-like bristles. This is called the axon. From the opposite end of the cell body sprout branch-like structures known as dendrites. By connecting the “branches” of their dendrites to the “root tips” of other neurons’ axons, nerve cells form networks, which can be as simple as the few connections involved in the knee-jerk reflex or as complex as those in the human brain.

Parker and her team found that embryonic nerve cells manufacture a well-known signaling enzyme called Atypical Protein Kinase C (aPKC) in two varieties: a full-length one and a truncated one. Both varieties compete to bind the same molecular partner, a protein called Par3. If the short form of aPKC pairs up with Par3, it tells the cell to grow a dendrite, and if the long one pairs up with Par3, it will make an axon instead.

When the researchers blocked the production of the short form, the nerve cell grew multiple axons and no dendrites. When they created an artificial abundance of the short form, dendrites formed at the expense of axons. UA undergraduate student Sophie Hapak performed many of the experiments revealing how the two isoforms compete for Par3.

"We show that wiring a neuronal circuit is much more complex than previously thought," said Ghosh. "The process has a built-in robustness that explicitly defines which part of the cell is ‘positive’ and which is ‘negative.’"

"In order to have a functioning neuronal circuit, you have to have receiving and sending ends," Parker said. "Initially, when a neuron is formed, it lacks the polarity it needs once it develops into a part of a circuit. The mechanism we discovered establishes that polarity."

"How the various brain regions are wired is the basis of emotion, memory and all cognitive functions," said Ghosh, who is a member of the UA’s BIO5 Institute. "Establishing neuronal polarity in single neurons is absolutely essential for neuronal circuits to form."

"If we understand this mechanism, we could think about methods to spur new axons after the original ones were severed in a traumatic spinal cord injury, for example," Ghosh said.

The findings defy conventional wisdom, which maintains that a developing neuron will make dendrites by default unless instructed by the long form of aPKC to make an axon instead. By cultivating and studying neurons just after they formed, Parker and her group found that both forms of aPKC, long and short, are initially distributed equally throughout the cell. These forms subsequently segregate into different parts of the cell as the neuron matures and establishes polarity.

Because the cells were isolated from rat brains and kept in culture, the researchers could demonstrate that no external clues from other cells are needed to instruct a developing neuron. Whether the establishment of polarity is a random process or whether other signals yet to be identified play a role in regulating the abundance of the two aPKC varieties is not known.

Hope for Spinal Cord Injuries

Laura Wong has coaxed damaged nerve cells to grow and send messages to the brain again

“An ailment not to be treated,” read the prescription for a spinal cord injury on an Egyptian papyrus in 1,700 B.C. Not much has changed in the intervening millennia. Despite decades of research, modern medicine has made little headway in its quest to reverse damage to the central nervous system.

That is not to say, however, that there isn’t a glimmer of hope. Laura Wong, an M.D./Ph.D. student in Professor Eric Frank’s molecular physiology lab at the Sackler School, has been able to coax damaged nerve cells known as sensory neurons to regenerate, growing as much as 10 times longer than previously documented. What’s more, the new neurons make organized connections with their counterparts inside the spinal cord and brain stem, ensuring information from the outside world paints an accurate picture inside the brain.

“All the regeneration in the world isn’t going to make any difference if they don’t reconnect. You’re still not going to get any function,” says Wong, who has worked since 2010 in Frank’s lab, which is trying to develop therapies for spinal cord injuries.

Her findings, which she presented at the annual meeting of the Society for Neuroscience in 2011 and 2012, shed light on the complex processes behind nerve cell growth and regeneration. If those results can be replicated in patients, it could prevent certain types of nerve damage and improve quality of life for some.

Going the Distance

Unlike tissues such as skin and bone, the cells of the central nervous system in an adult are notoriously resistant to healing. Not only does the supply of natural growth stimulants decline as we age, but the body also produces chemicals that discourage nerve cells from regenerating. Worse, the scar tissue that starts to form immediately after a spinal cord injury also contains compounds that hinder nerve cell growth.

Researchers in Frank’s lab have been seeking ways to either stimulate growth or block the mechanisms that inhibit nerve cell growth—or both—since 2005. Wong’s predecessor in the lab, Pamela Harvey, a 2009 graduate of the Sackler School, tested a synthetic version of a nerve cell growth factor, called artemin, on crushed sensory neurons that relay information from the hands, arms and shoulders to the brain.

The damage mimics a common injury called Erb’s palsy, which can occur when a baby’s shoulder gets caught behind the mother’s pelvis during labor and delivery, creating undue strain on nerves in the newborn’s neck. Riders thrown head first off a motorcycle or snowmobile can suffer similar injuries.

“Anytime the shoulder goes one way and the head and neck go the other, that’s when you see these injuries,” Wong says.

In earlier experiments, Harvey and Frank found that treating with artemin did indeed stimulate the sensory nerve fibers to regenerate and grow back into the spinal cord over the course of about six weeks. In her follow-up experiments, Wong showed that artemin could induce those nerve fibers to grow the 3- to 4-centimeter distance from there up to the brainstem, where the brain and the spinal cord meet. That’s a little more than an inch—or roughly 10 times longer than any other researchers have been able to demonstrate with artemin or any other growth factor.

“A lot of other researchers just haven’t seen this length,” notes Wong, who saw the artemin-induced growth occur over a period of three to six months.

That’s important, because while axons only have to grow across microscopic distances in a developing embryo, they would have to bridge much wider gaps—depending on the site of the injury—to heal a neural injury in an adult, Wong says. Nerves that extend from the spine to the foot or toe can reach lengths of about 60 centimeters, she adds.

But Wong’s artemin-treated nerve fibers achieved more than unprecedented growth. They also reestablished connections with correct regions in the brain stem, just as Harvey had seen the nerve cells do in the spinal cord. That is, the axons essentially plugged themselves back in just as they were prior to the injury, and, like an old-fashioned telephone switchboard, they sent the right messages to the right parts of the brain.

That’s crucial because should the sensory nerves that relay pain signals become crossed, for example, it could result in a patient feeling phantom pain or the sensation of pain from something that shouldn’t cause discomfort at all.

“With some other growth-promoting compounds you get regeneration, but you see those axons growing kind of willy-nilly,” says Wong. “You can see where it would be just as detrimental to have things wired incorrectly as it would be to have things not wired at all.”

Just a Start

Artemin isn’t a panacea for spinal cord injuries, Wong and Frank stress. To work its cellular magic, the compound must be administered within a day or two, and the sooner the better. Also, artemin promotes growth only in sensory neurons—and so far, only in rats—which means such growth wouldn’t improve motor function for someone who had been paralyzed by a spinal cord injury, for example.

But if the findings, which Wong presented at the Society for Neuroscience meetings in 2011 and 2012, prove applicable to humans, restoring sensation alone could still improve quality of life, even for those living with paralysis. Giving these people the ability to sense heat, cold and pain could help them avoid other accidental injuries, says Frank.

Wong hopes her work with sensory neurons will help unlock the secrets to promoting regeneration of other, more obstinate types of neurons in the brainstem and spinal cord. While she demonstrated that the sensory nerves plugged themselves back into the spinal cord precisely where they should have, it’s not clear how they did that.

Frank speculates that chemical cues guided the cells back into place. Should researchers be able to identify those cues, they potentially could use that knowledge to spark regeneration of other classes of neurons, such as motor neurons.

“There is hope—not proof—that even in humans these guidance molecules will persist into adulthood,” says Frank. “That means if we are able to get neurons to regenerate in patients, we might be able to make them go back to the right place. These experiments suggest we have some reason to believe it may work.”

Reversing Paralysis with a Restorative Gel

Some parts of the body, like the liver, can regenerate themselves after damage. But others, such as our nervous system, are considered either irreparable or slow to recover, leaving thousands with a lifetime of pain, limited mobility, or even paralysis.

Now a team of Tel Aviv University researchers, including Dr. Shimon Rochkind of TAU’s Sackler Faculty of Medicine and Tel Aviv Sourasky Medical Center and Prof. Zvi Nevo of TAU’s Department of Human Molecular Genetics and Biochemistry, has invented a method for repairing damaged peripheral nerves. Through a biodegradable implant in combination with a newly-developed Guiding Regeneration Gel (GRG) that increases nerve growth and healing, the functionality of a torn or damaged nerve could ultimately be restored.

This innovative project is now gaining international recognition. Its initial successes were reported recently at several renowned scientific congresses, including the World Federation of Neurological Societies and the European Neurological Society. And the therapy, already tested in animal models, is only a few years away from clinical use, says Dr. Rochkind.

Like healing in the womb

A nerve is like an electrical cable. When severed or otherwise damaged, power can no longer be transferred and the cable loses its functionality. Similarly, a damaged nerve loses the ability to transfer signals for movement and feeling through the nervous system.

But Dr. Rochkind and Prof. Nevo found a way to breach the gap. In their method, two severed ends of a damaged nerve are reconnected by implanting a soft, biodegradable tube, which serves as a bridge to help the nerve ends connect. The innovative gel which lines the inside of the tube nurtures nerve fibers’ growth, encouraging the nerve to reconnect the severed ends through the tube, even in cases with massive nerve damage, Dr. Rochkind says.

The key lies in the composition of the gel, the researchers say, which has three main components: anti-oxidants, which exhibit high anti-inflammatory activities; synthetic laminin peptides, which act as a railway or track for the nerve fibers to grow along; and hyaluronic acid, commonly found in the human fetus, which serves as a buffer against drying, a major danger for most implants. These components allow the nerve to heal the way a fetus does in the womb — quickly and smoothly.

Keeping cells safe for transplant

The implant has already been tested in animal models, and the gel by itself can be used as a stand-alone product, acting as an aid to cell therapy. GRG is not only able to preserve cells, it can support their survival while being used for therapy and transplantation, says Dr. Rochkind. When grown in the gel, cells show excellent development, as well as intensive fiber growth. This could have implications for the treatment of diseases such as Parkinson’s, for which researchers are actively exploring cell therapy as a potential solution.

Glial cells assist in the repair of injured nerves

Unlike the brain and spinal cord, the peripheral nervous system has an astonishing capacity for regeneration following injury. Researchers at the Max Planck Institute of Experimental Medicine in Göttingen have discovered that, following nerve damage, peripheral glial cells produce the growth factor neuregulin1, which makes an important contribution to the regeneration of damaged nerves.

From their cell bodies to their terminals in muscle or skin, neuronal extensions or axons in the peripheral nervous system are surrounded along their entire length by glial cells. These cells, which are known as Schwann cells, envelop the axons with an insulating sheath called myelin, which enables the rapid transmission of electrical impulses. Following injury to a peripheral nerve, the damaged axons degenerate. After a few weeks, however, they regenerate and are then recovered with myelin by the Schwann cells. For thus far unexplained reasons, however, the Schwann cells do not manage to regenerate the myelin sheaths completely. Thus the function of damaged nerves often remains permanently impaired and certain muscles remain paralysed in affected patients.

In a current research study, the scientists have succeeded in showing that the growth factor neuregulin1 supports nerve repair and the redevelopment of the myelin layer. This protein is usually produced by neurons and is localised on axons where it acts as an important signal for the maturation of Schwann cells and myelin formation. Because the axons rapidly degenerate after injury, the remaining Schwann cells lose their contact with the axons. They thus lack the neuregulin1 signal of the nervous fibres. “In the phase following nerve damage, in which the axons are missing, the Schwann cells must carry out many tasks without the help of axonal signals. If the Schwann cells cannot overcome this first major obstacle in the aftermath of nerve injury, the nerve cannot be adequately repaired,” explains Ruth Stassart, one of the study authors.

To prevent this, the Schwann cells themselves take over the production of the actual neuronal signal molecule. After nerve damage, they synthesise the neuregulin1 protein until the axons have grown again. With the help of genetically modified mice, the researchers working on this study were able to show that the neuregulin1 produced in Schwann cells is necessary for the new maturation of the Schwann cells and the regeneration of the myelin sheath after injury. “In mice that lack the neuregulin1 gene in their Schwann cells, the already incomplete nerve regeneration process is extensively impaired,” explains co-author Robert Fledrich.

The researchers would now like to examine in greater detail how the Schwann cells contribute to the complete repair of myelinated axons after nerve damage, so that this information can also be used for therapeutic purposes.