Researchers Uncover Key to Development of Peripheral Nervous System

Patients suffering from hereditary neuropathy may have hope for new treatment thanks to a Geisinger study that uncovered a key to the development of the peripheral nervous system.

In an article published today in the online medical journal Nature Communications, Geisinger researchers found that a protein present within immune system cells plays a larger role than previously thought in the development of the peripheral nervous system.

Nikolaos Tapinos, M.D., Ph.D., director of neurosurgery research and staff scientist at Geisinger’s Sigfried and Janet Weis Center for Research, said the findings could have implications in how hereditary neuropathy is treated. Hereditary neuropathy affects the peripheral nervous system, causing subtle symptoms such as muscle weakness, wasting and numbness that worsen over time.

“When the peripheral nervous system develops in utero, certain proteins control how the cells travel throughout the body to the proper locations,” Dr. Tapinos said. “Some of those proteins are already known, but this is the first time that the protein Lck has been identified as integral to this process.”

Lck, or lymphocyte-specific protein tyrosine kinase, is a protein that is found inside specialized cells of the immune system. Dr. Tapinos’ research found that Lck controls how cells called Schwann cells migrate across neurons throughout the peripheral nervous system.

Schwann cells function by creating the myelin sheath, the fatty covering that acts as an insulator around nerve fibers. In humans, the production of myelin begins in the 14th week of fetal development and continues through infancy and adolescence. When errors occur in the creation of myelin, hereditary neuropathy such as Charcot-Marie-Tooth disease (CMT), a motor and sensory neuropathy, can result.

“What we have found is that Lck is essentially the ‘switch’ that signals migration of the Schwann cells and production of the myelin sheath,” Dr. Tapinos said. “This finding sets the stage for further research into the specific molecular mechanisms that occur in order for this process to break down, and eventually toward developing treatments to prevent it.”

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A tangle of talents untangles neurons

Brown’s growing programs in brain science and engineering come together in the lab of Diane Hoffman-Kim. In a recent paper, her group employed techniques ranging from semiconductor-style circuit patterning to rat cell culture to optimize the growth of nerve cells for applications such as reconstructive surgery.

Two wrongs don’t make a right, they say, but here’s how one tangle can straighten out another.

Diane Hoffman-Kim, associate professor of medicine in the Department of Molecular Pharmacology, Physiology, and Biotechnology, is an affiliate of both Brown’s Center for Biomedical Engineering and the Brown Institute for Brain Science. Every thread of expertise woven through those multidisciplinary titles mattered in the Hoffman-Kim lab’s most recent paper, led by graduate student Cristina Lopez-Fagundo.

In research published online last month in Acta Biomaterialia, Hoffman-Kim and Lopez-Fagundo employed their neurophysiological knowledge and technological ingenuity to unravel a tangle of branching, tendrilous nerve cells, or neurons.

The scientist-engineers helped explain how neurons grow in new tissues in response to physical guideposts, called Schwann cells. Their paper also provided medical device makers with an overt demonstration of how to craft the best artificial Schwann cell implants in silicone to make neurons grow as straight as possible in a desired direction.

“If you’ve got an injury in your arm or your leg then you’d like to have proper reconnection so you can get function,” Hoffman-Kim said. “If it’s a small injury, your body does that fairly well in natural ways that largely depend on the Schwann cells. If the injury gets even just a little bit large then the Schwann cells can’t do it alone.”

Silicone Schwanns

Hoffman-Kim and Lopez-Fagundo did not invent the idea of creating an implant to direct neural growth through repaired or reattached tissues. Their clinical goal is to make that technology the best it can be by systematically studying neural growth on Schwann-like substrates. As a matter of basic science, they wanted to learn how neural growth proceeds.

Lopez-Fagundo, whom Hoffman-Kim recruited for her lab in 2008 when she applied to Brown after graduating from the University of Puerto Rico, started the research with rigorous measurements of Schwann cells in cell cultures of rat neural tissue — the cell size, their elliptical shape, and the average distance between any two, as well as the length and width of the “processes” or wispy extensions that connect them.

“We were able to deconstruct the topography of Schwann cells,” said Lopez-Fagundo. “We were then able to manipulate it into different designs to better understand the influence this topography has.”

They came up with six archetypal designs. One of them mimicked the somewhat messy real-world layout of Schwann cells but the other five were arranged in neat horizontal rows. In one the elliptical Schwann cell bodies were few and far between. In another they were densely packed and in another their spacing was the exact average of Lopez-Fagundo’s measurements. Another design had no “processes” to connect the ellipses and another had only processes but no ellipses.

Using Brown’s microfabrication facility, Lopez-Fagundo patterned their designs on silicon wafers (like those used to make computer chips) and then transferred them to silicone squares about a centimeter on a side so that the ellipses and processes were in raised relief on the silicone. Then they put each pattern in a cell culture of rat neurons and watched them as the neurons grew across each pattern of artificial Schwann cells. As a control for their experiment, they also cultured cells on unpatterned silicone squares.

All of the patterns encouraged some directed neuron growth compared to the random growth of neurons on the unpatterned squares, but clearly some patterns did better than others.

After 17 hours, the two best patterns were the ones with only processes and the one with average ellipse spacing. The natural replica pattern and the one with only ellipses fared the worst.

But by day five, new winners emerged: the patterns where the ellipses were farther than average and nearer than average. Hoffman-Kim said she was surprised that the nerve cells didn’t remain content to follow the straightforward pattern of plain horizontal tracks formed by the process-only pattern. Meanwhile, to some extent, the neurons grew the proper way even without a continuous track at all, for instance in the ellipse-only pattern.

Lopez-Fagundo puzzled over the question of why the ellipses, also called “soma,” matter even as the neurons clearly also grow along the processes.

“I asked myself that question a lot and it wasn’t until I sat at the computer and looked at the [time lapse] videos over and over,” Lopez-Fagundo said. “They use the soma as anchor points. They jump from soma to soma and use the long axis of the soma to guide themselves.”

It’s as if the neurons navigated most effectively when they had both roads (processes) and rest stops (ellipses or soma) where they could get their bearings.

And thus the neurons made their way along the artificially optimized straight and narrow. To the researchers, who also included co-authors Jennifer Mitchel, Talisha Ramchal, and Yu-Ting Dingle, the experiments were a triumph of how the meticulous analytical control afforded by engineering can demystify a complex biological phenomenon.

“Sometimes when I give lectures I say, ‘Biomedical engineers are control freaks and we consider that a compliment,’” Hoffman-Kim said.

New clues to causes of peripheral nerve damage

Anyone whose hand or foot has “fallen asleep” has an idea of the numbness and tingling often experienced by people with peripheral nerve damage. The condition also can cause a range of other symptoms, including unrelenting pain, stinging, burning, itching and sensitivity to touch.

Although peripheral neuropathies afflict some 20 million Americans, their underlying causes are not completely understood. Much research has focused on the breakdown of cellular energy factories in nerve cells as a contributing factor.

Now, new research at Washington University School of Medicine in St. Louis points to a more central role in damage to energy factories in other cells: Schwann cells, which grow alongside neurons and enable nerve signals to travel from the spinal cord to the tips of the fingers and toes.

The finding may lead to new therapeutic strategies to more effectively treat symptoms of this highly variable disorder, the scientists report March 6 in the journal Neuron.

“We found that a toxic substance builds up in Schwann cells that have disabled energy factories, leading to the same kind of nerve damage seen in patients with neuropathies,” says senior author Jeffrey Milbrandt, MD, PhD, the James S. McDonnell Professor of Genetics and head of the Department of Genetics. “Now, we’re evaluating whether drugs can block the buildup of that toxin, which could lead to a new treatment for the condition.”

The most common cause of peripheral neuropathy is diabetes, which accounts for about half of all cases. The condition also can occur in cancer patients treated with chemotherapy, which can damage nerves.

In the body, Schwann cells wrap tightly around nerve axons, the fibers that relay nerve signals. Graduate student and first author Andreu Viader and colleagues in Milbrandt’s lab studied Schwann cells in mice with genetically disabled mitochondria, or cellular energy factories. Under normal conditions, these mitochondria produce fuel and intermediates of energy metabolism that allow nerve cells to function.

The researchers showed that the crippled mitochondria activated a stress response in the Schwann cells. Instead of synthesizing fatty acids, a key component of Schwann cells, the cells burned fatty acids for fuel.

Over time, inefficient burning of fatty acids by the crippled mitochondria leads to a build up of acylcarnitines, a toxic substance, in the Schwann cells. The researchers found levels of acylcarnitines up to 100-fold higher in these mutant Schwann cells than in healthy Schwann cells.

And the bad news doesn’t end there. Eventually, the toxin leaks out of the Schwann cells and onto the nerve axons. Studying neurons in petri dishes, the researchers showed that acylcarnitines damage nerve axons and disrupt the ability of nerves to relay signals.

“The toxin leaking out of the Schwann cells and onto the adjacent nerve axons causes damage that results in pain, numbness, tingling and other symptoms,” Milbrandt says. “We think that is a likely mechanism to explain the degeneration of axons that is known to occur in peripheral neuropathies.”

The new research suggests that drugs that inhibit the buildup of acylcarnitines may block axonal degeneration. Milbrandt and his team now are evaluating the drugs in mice with disabled Schwann cells to see if they can slow or alleviate the decay of axons.

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.

Leprosy Bacteria Turn Nerve System Cells into Stem Cells

The study, carried out in mice, found that in the early stages of infection, M. leprae were able to protect themselves from the body’s immune system by hiding in the Schwann cells. Once the infection was fully established, the bacteria were able to convert the Schwann cells to become like stem cells.

Like typical stem cells, these cells were pluripotent, meaning they could then become other cell types, for instance muscle cells. This enabled M. leprae to spread to tissues in the body.

The study, published in the journal Cell, also shows that the bacteria-generated stem cells have unexpected characteristic. They can secrete specialized proteins – called chemokines – that attract immune cells, which in turn pick up the bacteria and spread the infection.

“We have found a new weapon in a bacteria’s armory that enables them to spread effectively in the body by converting infected cells to stem cells. Greater understanding of how this occurs could help research to diagnose bacterial infectious diseases, such as leprosy, much earlier,” said study lead author Prof Anura Rambukkana, Medical Research Council Center for Regenerative Medicine at the University of Edinburgh.

“This is very intriguing as it is the first time that we have seen that functional adult tissue cells can be reprogrammed into stem cells by natural bacterial infection, which also does not carry the risk of creating tumorous cells. Potentially you could use the bacteria to change the flexibility of cells, turning them into stem cells and then use the standard antibiotics to kill the bacteria completely so that the cells could then be transplanted safely to tissue that has been damaged by degenerative disease.”

Dr Rob Buckle, Head of Regenerative Medicine at the Medical Research Council Center for Regenerative Medicine at the University of Edinburgh, said: “this ground-breaking new research shows that bacteria are able to sneak under the radar of the immune system by hijacking a naturally occurring mechanism to ‘reprogramme’ cells to make them look and behave like stem cells. This discovery is important not just for our understanding and treatment of bacterial disease, but for the rapidly progressing field of regenerative medicine. In future, this knowledge may help scientists to improve the safety and utility of lab-produced pluripotent stem cells and help drive the development of new regenerative therapies for a range of human diseases, which are currently impossible to treat.”

The scientists believe mechanisms used by leprosy bacteria could exist in other infectious diseases. Knowledge of this newly discovered tactic used by bacteria to spread infection could help research to improve treatments and earlier diagnosis of infectious diseases.