Zebrafish study paves the way for new treatments for genetic disorder

Scientists from the University of Sheffield have paved the way for new treatments for a common genetic disorder thanks to pioneering research on zebrafish – an animal capable of mending its own heart.

Charcot Marie Tooth disease (CMT) is the most common genetic disorder affecting the nervous system. More than 20,000 people in the UK suffer from CMT, which typically causes progressive weakness and long-term pain in the feet, leading to walking difficulties. There is currently no cure for CMT.

A research project conducted at the Sheffield Institute for Translational Neuroscience (SITraN) and the MRC Centre for Developmental and Biomedical Genetics (CDBG) by Dr Andrew Grierson and his team has revealed that zebrafish could hold the key to finding new therapeutic approaches to treat the condition.

Dr Grierson said: “We have studied zebrafish with a genetic defect that causes CMT in humans. The fish develop normally, but once they reach adulthood they start to develop difficulties swimming.

"By looking at the muscles of these fish we have discovered that the problem lies with the connections between motor neurons and muscle, which are known to be essential for walking in humans and also swimming in fish."

CMT represents a group of neurodegenerative disorders typically characterised by demyelination (CMT1), a process which causes damage to the myelin sheaths that surround our neurons, or distal axon degeneration (CMT2) of motor and sensory neurons. The distal axon is the terminal where neurotransmitter packages within neurons are docked.

The majority of CMT2 cases are caused by mutations in mitofusin 2 (MFN2), which is an essential gene encoding a protein responsible for fusion of the mitochondrial outer membrane. Mitochondria are known as the cellular power plants because they generate most of the supply of adenosine triphosphate (ATP), which is used as a source of chemical energy.

Dr Grierson said: “Previous work on this disorder using mammalian models such as mice has been problematic, because the mitofusin genes are essential for embryonic development. Using zebrafish we were able to develop a model with an adult onset, progressive phenotype with predominant symptoms of motor dysfunction similar to CMT2.

"Motor neurons are the largest cells in our bodies, and as such they are highly dependent on a cellular transport system to deliver molecules through the long nerve cell processes which connect the spinal cord to our muscles. We already know that defects in the cellular transport system occur early in the development of diseases such as Alzheimer’s disease, Motor Neuron Disease and spastic paraplegia. Using our zebrafish model we have found that similar defects in transport are also a key part of the disease process in CMT."

Dr Grierson and his team are now seeking funding to identify new treatments for CMT using the zebrafish model. Because of their size and unique biology, zebrafish are ideal to be used in drug screens for the identification of new therapies for untreatable human conditions.

(Image courtesy: University College London)

Animal study shows promising path to prevent epilepsy

Duke Medicine researchers have identified a receptor in the nervous system that may be key to preventing epilepsy following a prolonged period of seizures.

Their findings from studies in mice, published online in the journal Neuron on June 20, 2013, provide a molecular target for developing drugs to prevent the onset of epilepsy, not just manage the disease’s symptoms.

"Unfortunately, there are no preventive therapies for any common disorder of the human nervous system – Alzheimer’s, Parkinson’s, schizophrenia, epilepsy – with the exception of blood pressure-lowering drugs to reduce the likelihood of stroke," said study author James O. McNamara, M.D., professor of neurobiology at Duke Medicine.

Epilepsy is a serious neurological disorder marked by recurring seizures. Temporal lobe epilepsy – where seizures occur in the region of the brain where memories are stored and language, emotions and senses are processed – is the most common form, and can be devastating. Because afflicted individuals have seizures that impair their awareness and may have associated behavioral problems, they may have difficulty with everyday activities, including holding a job or obtaining a driver’s license.

Conventional therapies to treat epilepsy address the disease’s symptoms by trying to reduce the likelihood of having a seizure. However, many people with temporal lobe epilepsy still have seizures despite taking these drugs.

"This study opens a promising new avenue of research into treatments that may prevent the development of epilepsy," said Vicky Whittemore, PhD, a program director at the National Institute of Neurological Disorders and Stroke, who oversees the grants that funded this study.

Retrospective studies of people with severe temporal lobe epilepsy reveal that many of them initially have an episode of prolonged seizures, known as status epilepticus. Status epilepticus is often followed by a period of seizure-free recovery before people start to experience recurring temporal lobe seizures.

In animal studies, inducing status epilepticus in an otherwise healthy animal can cause them to become epileptic. The prolonged seizures in status epilepticus are therefore thought to cause or importantly contribute to the development of epilepsy in humans.

"An important goal of this field has been to identify the molecular mechanism by which status epilepticus transforms a brain from normal to epileptic," said McNamara. "Understanding that mechanism in molecular terms would provide a target with which one could intervene pharmacologically, perhaps to prevent an individual from becoming epileptic."

Earlier research in epilepsy flagged a receptor in the nervous system called TrkB as a key player in transforming the brain from normal to epileptic. In the current study, McNamara and his colleagues sought to confirm if TrkB was important for status epilepticus-induced epilepsy.

Using an approach combining chemistry and genetic analyses, the researchers studied normal and genetically altered mice. The genetically altered mice were unique in that a drug, 1NMPP1, inhibited TrkB in their brains. If the drug stopped the genetically altered mice from becoming epileptic, this genetic approach would prove that inhibiting TrkB prevents the onset of epilepsy.

When the researchers caused status epilepticus in the animals, both the normal and genetically modified mice developed epilepsy. However, treatment with 1NMPP1 after the prolonged period of seizures prevented epilepsy in the genetically altered but not the normal mice.

"This demonstrated that it is possible to intervene following status epilepticus and prevent the animal from becoming epileptic," McNamara said.

Importantly, the researchers only administered treatment with 1NMPP1 for two weeks, which was sufficient to prevent epilepsy from developing in the mice when tested many weeks later. The results suggest that a preventive therapy may only need to be given for a limited period of time following the initial bout of prolonged seizures, not an individual’s entire life, which could prevent unnecessary side effects that come with long-term use of drugs.

In future studies, the researchers hope to determine the exact time window in which TrkB signaling needs to be repressed to prevent the onset of epilepsy. Long term, this research provides a molecular target for developing the first drugs to prevent epilepsy.

"This study provides a strong rationale for the development of selective inhibitors of TrkB signaling," said McNamara.

Study Points to Role of Nervous System in Arthritis

Arthritis is a debilitating disorder affecting one in 10 Canadians, with pain caused by inflammation and damage to joints.

Yet the condition is poorly managed in most patients, since adequate treatments are lacking – and the therapies that do exist to ease arthritis pain often cause serious side effects, particularly when used long-term. Any hope for developing more-effective treatments for arthritis relies on understanding the processes driving this condition.

A new study in the Journal of Neuroscience by researchers at McGill University adds to a growing body of evidence that the nervous system and nerve-growth factor (NGF) play a major role in arthritis. The findings also support the idea that reducing elevated levels of NGF – a protein that promotes the growth and survival of nerves, but also causes pain — may be an important strategy for developing treatment of arthritis pain.

Using an approach established by arthritis researchers elsewhere, the McGill scientists examined inflammatory arthritis in the ankle joint of rats. In particular, they investigated changes in the nerves and tissues around the arthritic joint, by using specific markers to label the different types of nerve fibres and allow them to be visualized with a fluorescence microscope.

Normally, sympathetic nerve fibres regulate blood flow in blood vessels. Following the onset of arthritis in the rats, however, these fibres began to sprout into the inflamed skin over the joint and wrap around the pain-sensing nerve fibres instead. More sympathetic fibres were detected in the arthritic joint tissues, as well.

The results also showed a higher level in the inflamed skin of NGF – mirroring the findings of human studies that have shown considerable increases in NGF levels in arthritis patients.

To investigate the role of these abnormal sympathetic fibres, the McGill researchers used an agent to block the fibres’ function. They found that this reduced pain-related behaviour in the animals.

“Our findings reinforce the idea that there is a neuropathic component to arthritis, and that sympathetic nerve fibres play a role in increasing the pain,” said McGill doctoral student Geraldine Longo, who co-authored the paper with Prof. Afredo Ribeiro-da-Silva and postdoctoral fellow Maria Osikowicz.

“We are currently using drugs to prevent the production of elevated levels of NGF in arthritic rats; we hope that our research will serve as a basis for the development of a new treatment for arthritis in the clinic”, said Prof. Ribeiro-da-Silva.

Testing method promising for spinal cord injuries, multiple sclerosis

A medical test previously developed to measure a toxin found in tobacco smokers has been adapted to measure the same toxin in people suffering from spinal cord injuries and multiple sclerosis, offering a potential tool to reduce symptoms.

The toxin, called acrolein, is produced in the body after nerve cells are injured, triggering a cascade of biochemical events thought to worsen the injury’s severity. Acrolein (pronounced a-KRO-le-an) also may play an important role in multiple sclerosis and other conditions.

Because drugs already exist to reduce the concentration of acrolein in the body, being able to detect and measure it non-invasively represents a potential treatment advance, said Riyi Shi (pronounced Ree Shee), a professor of neuroscience and biomedical engineering in Purdue University’s Department of Basic Medical Sciences, School of Veterinary Medicine, Center for Paralysis Research and Weldon School of Biomedical Engineering.

"If the acrolein level is high it needs to be reduced, and we already have effective acrolein removers to do so," Shi said. "Reducing or removing acrolein may lessen the severity of symptoms in people who have nerve damage, but there has not been a practical way to monitor acrolein levels in nervous system trauma and diseases."

The toxin is present in tobacco smoke and air pollutants. A method had been developed previously to detect and measure acrolein in the urine of smokers, but it has not been used in people suffering from conditions in which the body produces acrolein internally.

"Based on this method, it was revealed that acrolein is significantly elevated in smokers and decreases following the cessation of cigarette smoke," Shi said. "However, such a method has not been widely used for conditions in which acrolein is elevated due to central nervous system damage or disease."

The researchers tested the method in laboratory animals.

"We wanted to see if higher levels of acrolein corresponds to greater severity of spinal cord injury, and the answer is yes," said Shi, who is working with Bruce Cooper, director of the Metabolite Profiling Facility in the Bindley Bioscience Center of Purdue’s Discovery Park. "This means reducing acrolein may help to control symptoms."

New findings are detailed in a research paper that recently appeared online in the Journal of Neurotrauma. The paper, which also will appear in an upcoming print edition of the journal, was authored by doctoral students Lingxing Zheng, Jonghyuck Park, Michael Walls and Melissa Tully; Amber Jannasch, laboratory manager of the Metabolite Profiling Facility; and Cooper and Shi.

The method does not detect acrolein directly but determines the presence of a byproduct, or metabolite, of acrolein in the urine. The metabolite is a chemical compound called N-acetyl-S-3-Hydroxypropylcysteine, or 3-HPMA.

"Acrolein is very volatile, so it doesn’t remain stable long enough to monitor, but one molecule of acrolein will make one molecule of 3-HPMA, which is very stable in urine," Shi said.

Laboratory rats were injected with different doses of acrolein, and findings showed that the detection technique is able to accurately measure these differences in acrolein concentration in the urine. The technique might one day be performed routinely in a doctor’s office.

"The non-invasive nature of measuring 3-HPMA concentrations in urine allows for long-term monitoring of acrolein in the same animal and ultimately in human clinical studies," Shi said.

Two drugs have been shown to be effective in reducing acrolein levels in the body: hydralazine and phenelzine, which have been approved by the U.S. Food and Drug Administration for hypertension and depression, respectively.

The testing method could be used in conjunction with other measures to test patients for the progress of spinal cord disease.

"Nervous system trauma and diseases are like many other illnesses: A disease-associated marker can be critical for making a diagnosis, a therapeutic selection and a treatment evaluation," Shi said. "Therefore, determination of acrolein levels gives you more assurance that you have an intense biochemical imbalance and biochemical damage and that you should use an acrolein scavenger as a treatment. We used different levels of hydralazine to see if it causes a dose-dependent reduction of 3-HPMA and found that, in fact, it did. This shows that this method is capable of monitoring the decrease of acrolein through treatment with acrolein-removing medications."

Acrolein damages mitochondria, which provide energy for cells, and in multiple sclerosis compromises the myelin sheath surrounding a nerve cell’s axon, preventing nerves from properly conducting electrical impulses. The toxin has a possible role in other diseases, including Alzheimer’s disease, cancer and atherosclerosis.

"Due to widespread involvement of acrolein in the body, the benefits of this study have the potential to significantly enhance human health," Shi said. "For example, there is evidence that heightened levels of acrolein could diminish an individual’s ability to recover fully from stroke and cancer."

In laboratory animals, hydralazine has been shown to delay onset of multiple sclerosis for several days, which could mean several years in humans. Tests with animals also suggests the drug could help to reduce the most severe symptoms once the disease has progressed.

Acrolein has been found to be elevated by about 60 percent in the spinal cord tissues of mice with a disease similar to multiple sclerosis. The toxin causes harm by reacting with the proteins and lipids that make up cells, including neurons.

Motor neurons like this one, found in the crab Cancer borealis, underlie the walking, swimming, breathing, flying and other rhythmic behaviors found in most creatures, including humans.

Eve Marder wins 2013 Gruber Neuroscience Prize

Award recognizes ‘the best neuroscience research being done anywhere’

The Gruber Foundation today awarded its 2013 neuroscience prize to Eve Marder ’69, a pioneering researcher who has dedicated her career to understanding the nervous system’s basic functions. The Victor and Gwendolyn Beinfield Professor of Neuroscience at Brandeis, Marder studies a relatively simple network of some 30 large neurons found in the gut of lobsters and crabs — a small yet elegant window into humans’ unfathomably rich nervous system, home to billions of neurons and trillions of interconnections.

The $500,000 prize recognizes and rewards “the best [neuroscience] work being done anywhere in the world,” according to the Gruber Foundation website. 

"Eve Marder has made a number of remarkable and groundbreaking discoveries that have fundamentally changed our understanding of how neural circuits operate and produce behavior," says Carol Barnes, chair of the selection advisory board to the Neuroscience Prize. "She has also been an exceptional leader outside the laboratory, working tirelessly to bring people together to improve scientific research, policy, and education."

Marder’s singular contributions to neuroscience through her use of crustaceans — in a field heavily dominated by scientists using vertebrate model organisms, chiefly rodents — have helped define how we think about neurons and their astounding capabilities. 

Despite not practicing “consensus” science — Marder avoids the well-trodden path of established modes of inquiry, such as working in vertebrates — she has received numerous accolades, including election to the National Academy of Sciences and to the helm of the Society for Neuroscience, both in 2007.

“I’m a maverick within a conservative framework — I obey carefully the rules of scientific rigor and discipline,” says Marder, who began her freshman year at Brandeis thinking she would major in politics. By her senior year, enthralled with the emerging field of neuroscience, she applied to graduate school while some of her friends made their plans to join the counterculture.

As a graduate student at the University of California, San Diego, in the early 1970s, Marder began studying the stomatogastric nervous system of the West Coast spiny lobster, Panulirus interruptus. The stomatogastric nervous system, which controls the motion of the gut, is an example of a central pattern generator. These circuits generate organized and repetitive motor patterns that also underlie walking, swimming, flying, breathing and many other rhythmic behaviors that creatures from earthworms to humans take for granted. 

The big questions Marder has asked throughout her career attempt to understand the fundamental nature of neuronal circuit operation. In a Brandeis lab staffed by post-docs, graduate students and undergraduates, she’s helped advance basic tenets of neuroscience while continuing to refine several related lines of inquiry. 

Early in Marder’s Brandeis career, her lab demonstrated that neuromodulatory substances such as dopamine, serotonin and neuropeptides can alter circuit performance so that the same group of neurons can produce a variety of behaviors. Her research has helped reshape the way scientists think about conditions like depression, now believed to stem from imbalances in neuromodulation. 

Later, her lab studied how neurons and networks maintain stable network performance despite the ongoing turnover of the membrane proteins that give neurons their characteristic electrical properties. Most recently, her lab is studying animal-to-animal variability in neuronal properties. How much variability in circuit function is there between animals even as they respond similarly to changes in hormones or temperature?

“I’m always looking for the things we can study more effectively than someone working in a large nervous system,” explains Marder. “I don’t want to work on problems that someone else can do better.”

Awarded by a distinguished panel of experts following an international nomination process, the Gruber Foundation neuroscience prize is a humbling honor, Marder says. It is also recognition that great science requires both intellectual risk-taking and persistence. 

Marder plans to celebrate, just not over a fancy lobster dinner. She gave up eating crustaceans long ago.

Scientists Discover Molecule Triggers Sensation of Itch

Scientists at the National Institutes of Health report they have discovered in mouse studies that a small molecule released in the spinal cord triggers a process that is later experienced in the brain as the sensation of itch.

The small molecule, called natriuretic polypeptide b (Nppb), streams ahead and selectively plugs into a specific nerve cell in the spinal cord, which sends the signal onward through the central nervous system. When Nppb or its nerve cell was removed, mice stopped scratching at a broad array of itch-inducing substances. The signal wasn’t going through.

Because the nervous systems of mice and humans are similar, the scientists say a comparable biocircuit for itch likely is present in people. If correct, this start switch would provide a natural place to look for unique molecules that can be targeted with drugs to turn off the sensation more efficiently in the millions of people with chronic itch conditions, such eczema and psoriasis.

The paper, published online in the journal Science, also helps to solve a lingering scientific issue. “Our work shows that itch, once thought to be a low-level form of pain, is a distinct sensation that is uniquely hardwired into the nervous system with the biochemical equivalent of its own dedicated land line to the brain,” said Mark Hoon, Ph.D., the senior author on the paper and a scientist at the National Institute of Dental and Craniofacial Research, part of the National Institutes of Health.

Hoon said his group’s findings began with searching for the signaling components on a class of nerve cells, or neurons, that contain a molecule called TRPV1. These neurons, with their long nerve fibers extending into the skin, muscle, and other tissues, help to monitor a range of external conditions, from extreme temperature changes to detecting pain.

Yet little is known about how these neurons recognize the various sensory inputs and, like sorting mail, know how to route them correctly to the appropriate pathway to the brain.

To fill in more of the details, Hoon said his laboratory identified in mice some of the main neurotransmitters that TRPV1 neurons produce. A neurotransmitter is a small molecule that neurons selectively release when stimulated, like a quick pulse of water from a faucet, to communicate sensory signals to other nerve cells.

The scientists screened the various neurotransmitters, including Nppb, to see which ones corresponded with transmitting sensation.

“We tested Nppb for its possible role in various sensations without success,” said Santosh Mishra, lead author on the study and a researcher in the Hoon laboratory. “When we exposed the Nppb-deficient mice to several itch-inducing substances, it was amazing to watch. Nothing happened. The mice wouldn’t scratch.”

Further experiments established that Nppb was essential to initiate the sensation of itch, known clinically as pruritus. Equally significant, the molecule was necessary to respond to a broad spectrum of pruritic substances. Previous research had suggested that a common start switch for itch would be unlikely, given the myriad proteins and cell types that seemed to be involved in processing the sensation.

Hoon and Mishra turned to the dorsal horn, a junction point in the spine where sensory signals from the body’s periphery are routed onward to the brain. Within this nexus of nerve connections, they looked for cells that expressed the receptor to receive the incoming Nppb molecules.

“The receptors were exactly in the right place in the dorsal horn,” said Hoon, the receptor being the long-recognized protein Npra. “We went further and removed the Npra neurons from the spinal cord. We wanted to see if their removal would short-circuit the itch, and it did.”

Hoon said this experiment added another key piece of information. Removing the receptor neurons had no impact on other sensory sensations, such as temperature, pain, and touch. This told them that the connection forms a dedicated biocircuit to the brain that conveys the sensation of itch.

But the scientists had stepped into a conundrum. Previous reports had suggested that another neurotransmitter called GRP might initiate itch. If that wasn’t the case, where did GRP fit into the process?

They tested the receptor neurons that express GRP, finding the previous reports were correct about this molecule relaying the signal to the central nervous system. GRP just enters the picture after Nppb already has set the sensation in motion.

Based on these findings, Nppb would seem to be an obvious first target to control itch. But that’s not necessarily the case. Nppb also is used in the heart, kidneys, and other parts of the body, so attempts to control the neurotransmitter in the spine has the potential to cause unwanted side effects.

“The larger scientific point remains,” said Hoon. “We have defined in the mouse the primary itch-initiating neurons and figured out the first three steps in the pruritic pathway. Now the challenge is to find similar biocircuitry in people, evaluate what’s there, and identify unique molecules that can be targeted to turn off chronic itch without causing unwanted side effects. So, this is a start, not a finish.”

(Image: GETTY)

‘Brainbow,’ version 2.0: Researchers refine breakthrough system for producing images of brain, nervous system

The breakthrough technique that allowed scientists to obtain one-of-a-kind, colorful images of the myriad connections in the brain and nervous system is about to get a significant upgrade.

A group of Harvard researchers, led by Joshua Sanes, the Jeff C. Tarr Professor of Molecular and Cellular Biology and Paul J. Finnegan Family Director, Center for Brain Science, and Jeff Lichtman, the Jeremy R. Knowles Professor of Molecular and Cellular Biology and Santíago Ramón y Cajal Professor of Arts and Sciences, has made a host of technical improvements in the “Brainbow” imaging technique. Their work is described in a May 5 paper in Nature Methods.

First described in 2007, the system combines three fluorescent proteins — one red, one blue, and one green — to label different cells with as many as 90 colors. By studying the resulting images, researchers were able to begin to understand how the millions of neurons in the brain are connected.

“‘Brainbow’ generated beautiful images of a kind we had never been able to obtain before, but it was difficult in some ways,” said Sanes, who also serves as director of the Center for Brain Science.

“These modifications aim to overcome some of the more problematic features of the original genetic constructs,” Lichtman said. “Lead author Dawen Cai, a research associate in our labs, worked hard and creatively to find ways to make the ‘Brainbow’ colors brighter, more variable, and useable in situations where the original gene constructs were hard to implement. Our first look at these animals suggests that these improvements are fantastic.”

Among the challenges faced by researchers using the original method, Sanes said, was the chance that certain colored proteins would bleach out faster than others.

“If one color bleaches faster than the others, you start with a ‘Brainbow,’ but by the time you’re done imaging, you might just have a ‘blue-bow,’ because the red and yellow bleach too fast,” he said.

Sanes said that some colors also were too dim, causing problems in the imaging process, while in other cases the protein didn’t fill the whole neuron evenly enough, or there was an overabundance of a certain color in an image.

“What we decided to do was to make the next generation of ‘Brainbow,’” Sanes said. “We systematically set out to look at these problems. We looked at a whole range of fluorescent proteins to find the ones that were brightest and wouldn’t bleach as much, and we developed new transgenic methods to avoid the predominance of a particular color.”

The researchers also explored new ways to create “Brainbow” images, including using viruses to introduce fluorescent proteins into cells.

The advantage of the new technique, Sanes said, is it offers researchers the chance to target certain parts of the brain and better understand how neurons radiate out to connect with other brain regions. Ultimately, he said, he hopes that other researchers are able to apply the techniques outlined in the paper in the same way that they expanded on the first “Brainbow” method.

“People adapted the method to study a number of interesting questions in other tissues to examine cellular relationships and cell lineages in kidney and skin cells,” he said. “It was also used to examine the nervous system in animals like zebrafish and C. elegans. With these new tools, I think we’ve taken the next step.”