Muscle-controlling Neurons Know When They Mess Up

Whether it is playing a piano sonata or acing a tennis serve, the brain needs to orchestrate precise, coordinated control over the body’s many muscles. Moreover, there needs to be some kind of feedback from the senses should any of those movements go wrong. Neurons that coordinate those movements, known as Purkinje cells, and ones that provide feedback when there is an error or unexpected sensation, known as climbing fibers, work in close concert to fine-tune motor control.   

A team of researchers from the University of Pennsylvania and Princeton University has now begun to unravel the decades-spanning paradox concerning how this feedback system works.

At the heart of this puzzle is the fact that while climbing fibers send signals to Purkinje cells when there is an error to report, they also fire spontaneously, about once a second. There did not seem to be any mechanism by which individual Purkinje cells could detect a legitimate error signal from within this deafening noise of random firing. 

Using a microscopy technique that allowed the researchers to directly visualize the chemical signaling occurring between the climbing fibers and Purkinje cells of live, active mice, the Penn team has for the first time shown that there is a measurable difference between “true” and “false” signals.

This knowledge will be fundamental to future studies of fine motor control, particularly with regards to how movements can be improved with practice. 

The research was conducted by Javier Medina, assistant professor in the Department of Psychology in Penn’s School of Arts and Sciences, and Farzaneh Najafi, a graduate student in the Department of Biology. They collaborated with postdoctoral fellow Andrea Giovannucci and associate professor Samuel S. H. Wang of Princeton University.

It was published in the journal Cell Reports.

The cerebellum is one of the brain’s motor control centers. It contains thousands of Purkinje cells, each of which collects information from elsewhere in the brain and funnels it down to the muscle-triggering motor neurons. Each Purkinje cell receives messages from a climbing fiber, a type of neuron that extends from the brain stem and sends feedback about the associated muscles. 

“Climbing fibers are not just sensory neurons, however,” Medina said. “What makes climbing fibers interesting is that they don’t just say, ‘Something touched my face’; They say, ‘Something touched my face when I wasn’t expecting it.’ This is something that our brains do all the time, which explains why you can’t tickle yourself. There’s part of your brain that’s already expecting the sensation that will come from moving your fingers. But if someone else does it, the brain can’t predict it in the same way and it is that unexpectedness that leads to the tickling sensation.”

Not only does the climbing fiber feedback system for unexpected sensations serve as an alert to potential danger — unstable footing, an unseen predator brushing by — it helps the brain improve when an intended action doesn’t go as planned.    

“The sensation of muscles that don’t move in the way the Purkinje cells direct them to also counts as unexpected, which is why some people call climbing fibers ‘error cells,’” Medina said. “When you mess up your tennis swing, they’re saying to the Purkinje cells, ‘Stop! Change! What you’re doing is not right!’ That’s where they help you learn how to correct your movements.

“When the Purkinje cells get these signals from climbing fibers, they change by adding or tweaking the strength of the connections coming in from the rest of the brain to their dendrites. And because the Purkinje cells are so closely connected to the motor neurons, the changes to those synapses are going to result in changes to the movements that Purkinje cell controls.”

This is a phenomenon known as neuroplasticity, and it is fundamental for learning new behaviors or improving on them. That new neural pathways form in response to error signals from the climbing fibers allows the cerebellum to send better instructions to motor neurons the next time the same action is attempted.

The paradox that faced neuroscientists was that these climbing fibers, like many other neurons, are spontaneously activated. About once every second, they send a signal to their corresponding Purkinje cell, whether or not there were any unexpected stimuli or errors to report.

“So if you’re the Purkinje cell,” Medina said, “how are you ever going to tell the difference between signals that are spontaneous, meaning you don’t need to change anything, and ones that really need to be paid attention to?”

Medina and his colleagues devised an experiment to test whether there was a measurable difference between legitimate and spontaneous signals from the climbing fibers. In their study, the researchers had mice walk on treadmills while their heads were kept stationary. This allowed the researchers to blow random puffs of air at their faces, causing them to blink, and to use a non-invasive microscopy technique to look at how the relevant Purkinje cells respond.

The technique, two-photon microscopy, uses an infrared laser and a reflective dye to look deep into living tissue, providing information on both structure and chemical composition. Neural signals are transmitted within neurons by changing calcium concentrations, so the researchers used this technique to measure the amount of calcium contained within the Purkinje cells in real time.

Because the random puffs of air were unexpected stimuli for the mice, the researchers could directly compare the differences between legitimate and spontaneous signals in the eyelid-related Purkinje cells that made the mice blink.

“What we have found is that the Purkinje cell fills with more calcium when its corresponding climbing fiber sends a signal associated with that kind of sensory input, rather than a spontaneous one,” Medina said. “This was a bit of a surprise for us because climbing fibers had been thought of as ‘all or nothing’ for more than 50 years now.”

The mechanism that allows individual Purkinje cells to differentiate between the two kinds of climbing fiber signals is an open question. These signals come in bursts, so the number and spacing of the electrical impulses from climbing fiber to Purkinje cell might be significant. Medina and his colleagues also suspect that another mechanism is at play: Purkinje cells might respond differently when a signal from a climbing fiber is synchronized with signals coming elsewhere from the brain.   

Whether either or both of these explanations are confirmed, the fact that individual Purkinje cells are able to distinguish when their corresponding muscle neurons encounter an error must be taken into account in future studies of fine motor control. This understanding could lead to new research into the fundamentals of neuroplasticity and learning.    

“Something that would be very useful for the brain is to have information not just about whether there was an error but how big the error was — whether the Purkinje cell needs to make a minor or major adjustment,” Medina said. “That sort of information would seem to be necessary for us to get very good at any kind of activity that requires precise control. Perhaps climbing fiber signals are not as ‘all-or-nothing’ as we all thought and can provide that sort of graded information”

Image caption: New details about how motor neurons die in ALS have been uncovered by a new cell-culture system that combines spinal cord or brain cells from ALS patients with human motor neurons. The culture system shows that patient astrocytes (shown here with a blue-stained nucleus) release a toxin that kills motor neurons via a recently discovered process described as a “controlled cellular explosion.” Image: Diane Re.

Toxin from Brain Cells Triggers Neuron Loss in Human ALS Model

In most cases of amyotrophic lateral sclerosis (ALS), or Lou Gehrig’s disease, a toxin released by cells that normally nurture neurons in the brain and spinal cord can trigger loss of the nerve cells affected in the disease, Columbia researchers reported today in the online edition of the journal Neuron.

The toxin is produced by star-shaped cells called astrocytes and kills nearby motor neurons. In ALS, the death of motor neurons causes a loss of control over muscles required for movement, breathing, and swallowing. Paralysis and death usually occur within 3 years of the appearance of first symptoms.

The report follows the researchers’ previous study, which found similar results in mice with a rare, genetic form of the disease, as well as in a separate study from another group that used astrocytes derived from patient neural progenitor cells. The current study shows that the toxins are also present in astrocytes taken directly from ALS patients.

“I think this is probably the best evidence we can get that what we see in mouse models of the disease is also happening in human patients,” said the study’s senior author, Serge Przedborski, MD, PhD, the Page and William Black Professor of Neurology (in Pathology and Cell Biology), Vice Chair for Research in the department of Neurology, and co-director of Columbia’s Motor Neuron Center.

The findings also are significant because they apply to the most common form of ALS, which affects about 90 percent of patients. Scientists do not know why ALS develops in these patients; the other 10 percent of patients carry one of 27 genes known to cause the disease.

“Now that we know that the toxin is common to most patients, it gives us an impetus to track down this factor and learn how it kills the motor neurons,” Dr. Przedborski said. “Its identification has the potential to reveal new ways to slow down or stop the destruction of the motor neurons.”

In the study, Dr. Przedborski and study co-authors Diane Re, PhD, and Virginia Le Verche, PhD, associate research scientists, removed astrocytes from the brain and spinal cords of six ALS patients shortly after death and placed the cells in petri dishes next to healthy motor neurons. Because motor neurons cannot be removed from human subjects, they had been generated from human embryonic stem cells in the Project A.L.S./Jenifer Estess Laboratory for Stem Cell Research, also at CUMC.

Within two weeks, many of the motor neurons had shrunk and their cell membranes had disintegrated; about half of the motor neurons in the dish had died. Astrocytes removed from people who died from causes other than ALS had no effect on the motor neurons. Nor did other types of cells taken from ALS patients.

The researchers confirmed that the cause of the motor neurons’ death was a toxin released into the environment by immersing healthy motor neurons in the astrocytes’ culture media. The presence of the media, even without astrocytes, killed the motor neurons.

 How the Toxin Triggers Motor Neuron Death

The researchers have not yet identified the toxin released by the astrocytes. But they did discover the nature of the neuronal death process triggered by the toxin.The toxin triggers a biochemical cascade in the motor neurons that essentially causes them to undergo a controlled cellular explosion.

Drs. Przedborski, Re, and Le Verche found that they could prevent astrocyte-triggered motor neuron death by inhibiting one of the key components of this molecular cascade.

These findings may lead to a way to prevent motor neuron death in patients and potentially prolong life. But the therapeutic potential of such inhibition is far from clear. “For example, we don’t know if this would leave patients with living but dysfunctional neurons,” Dr. Przedborski said. The researchers are now testing the idea of inhibition in animal models of ALS.

New Human Cell Model of ALS Will Speed Identification of Potential Therapies

The development of new therapies for ALS has been disappointing, with more than 30 clinical trials ending with no new treatments since the 1995 FDA approval of riluzole.

The lack of progress may be partly because animal models used to study ALS do not completely recreate the human disease. The new all-human cell model of ALS created for the current study may improve scientists’ ability to identify useful drug targets, particularly for the most common form of the disease.

“Although there are many neurodegenerative disorders, only for a handful do we have access to a simplified model that is relevant to the disease and can therefore potentially be used for high-throughput drug screening. So this model is quite special,” Dr. Przedborski said. “Here we have a spontaneous disease phenotype triggered by the relevant tissue that causes human illness. That’s one important thing. The other important thing is that this model is derived entirely from human elements. This is probably the closest, most natural model of human ALS that we can get in a dish.”

CC to the brain: How neurons control fine motor behavior of the arm

Motor commands issued by the brain to activate arm muscles take two different routes. As the research group led by Professor Silvia Arber at the Basel University Biozentrum and the Friedrich Miescher Institute for Biomedical Research has now discovered, many neurons in the spinal cord send their instructions not only towards the musculature, but at the same time also back to the brain via an exquisitely organized network. This dual information stream provides the neural basis for accurate control of arm and hand movements. These findings have now been published in “Cell”.

Movement is a fundamental capability of humans and animals, involving the highly complex interplay of brain, nerves and muscles. Movements of our arms and hands, in particular, call for extremely precise coordination. The brain sends a constant stream of commands via the spinal cord to our muscles to execute a wide variety of movements. This stream of information from the brain reaches interneurons in the spinal cord, which then transmit the commands via further circuits to motor neurons innervating muscles. The research group led by Silvia Arber at the Biozentrum of the University of Basel and the Friedrich Miescher Institute for Biomedical Research has now elucidated the organization of a second information pathway taken by these commands.

cc to the brain: one command – two directions

The scientists showed that many interneurons in the mouse spinal cord not only transmit their signals via motor neurons to the target muscle, but also simultaneously send a copy of this information back to the brain. Chiara Pivetta, first author of the publication, explains: “The motor command to the muscle is sent in two different directions – in one direction, to trigger the desired muscular contraction and in the other, to inform the brain that the command has actually been passed on to the musculature.” In analogy to e‑mail transmission, the information is thus not only sent to the recipient but also to the original requester.

Information to brainstem nucleus segregated by function

What happens to the information sent by spinal interneurons to the brain? As Arber’s group discovered, this input is segregated by function and spatially organized within a brainstem nucleus. Information from different types of interneurons thus flows to different areas of the nucleus. For example, spinal information that will influence left-right coordination of a movement is collected at a different site than information affecting the speed of a movement.

Fine motor skills supported by dual information stream

Arber comments: “From one millisecond to the next, this extremely precise feedback ensures that commands are correctly transmitted and that – via the signals sent back to the brain from the spinal cord – the resulting movement is immediately coordinated with the brain and adjusted.” Interestingly, the scientists only observed this kind of information flow to the brain for arm, but not for leg control. “What this shows,” says Arber, “is that this information pathway is most likely important for fine motor skills. Compared to the leg, movements of our arm and especially our hands have to be far more precise. Evidently, our body can only ensure this level of accuracy in motor control with constant feedback of information.”

In further studies, Silvia Arber’s group now plans to investigate what happens if the flow of information back to the brain is disrupted in specific ways. Since some interneurons facilitate and others inhibit movement, such studies could provide additional insights into the functionality of circuits controlling movement.

Image caption: MMP-9 controls onset of paralysis in ALS mice. Sections of muscle stained for nerve (green) and muscle (red); nerve-muscle contacts appear yellow. In the SOD1 mouse, muscles that move the eye (left) retain nerve contacts and are active. Fast leg muscles (center) in the same animal lose nerve contacts (red stain only) and become paralyzed. Fast muscles from which MMP-9 has been genetically removed (right) retain their nerve contacts, and therefore muscle function, for nearly 3 months longer. This suggests that inhibiting MMP-9 in human patients with ALS should be beneficial. Credit: The Henderson Lab/Columbia University Medical Center.

Study Identifies Gene Tied to Motor Neuron Loss in ALS

Columbia University Medical Center (CUMC) researchers have identified a gene, called matrix metalloproteinase-9 (MMP-9), that appears to play a major role in motor neuron degeneration in amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease. The findings, made in mice, explain why most but not all motor neurons are affected by the disease and identify a potential therapeutic target for this still-incurable neurodegenerative disease. The study was published today in the online edition of the journal Neuron.

“One of the most striking aspects of ALS is that some motor neurons—specifically, those that control eye movement and eliminative and sexual functions—remain relatively unimpaired in the disease,” said study leader Christopher E. Henderson, PhD, the Gurewitsch and Vidda Foundation Professor of Rehabilitation and Regenerative Medicine, professor of pathology & cell biology and neuroscience (in neurology), and co-director of Columbia’s Motor Neuron Center. “We thought that if we could find out why these neurons have a natural resistance to ALS, we might be able to exploit this property and develop new therapeutic options.”

To understand why only some motor neurons are vulnerable to ALS, the researchers used DNA microarray profiling to compare the activity of tens of thousands of genes in neurons that resist ALS (oculomotor neurons/eye movement and Onuf’s nuclei/continence) with neurons affected by ALS (lumbar 5 spinal neurons/leg movement). The neurons were taken from normal mice.

“We found a number of candidate ‘susceptibility’ genes—genes that were expressed only in vulnerable motor neurons. One of those genes, MMP-9, was strongly expressed into adulthood. That was significant, as ALS is an adult-onset disease,” said co-lead author Krista J. Spiller, a former graduate student in Dr. Henderson’s laboratory who is now a postdoctoral fellow at the University of Pennsylvania. The other co-lead author is Artem Kaplan, a former MD-PhD student in the lab who is now a neurology resident at NewYork-Presbyterian Hospital/Columbia University Medical Center.

In a follow-up experiment, the researchers confirmed that the product of MMP-9, MMP-9 protein, is present in ALS-vulnerable motor neurons, but not in ALS-resistant ones. Further, the researchers found that MMP-9 can be detected not just in lumbar 5 neurons, but also in other types of motor neurons affected by ALS. “It was a perfect correlation.” said Dr. Henderson. “In other words, having MMP-9 is an absolute predictor that a motor neuron will die if the disease strikes, at least in mice.”

Taking a closer look at the groups of vulnerable motor neurons, the researchers found differences in MMP-9 expression at the single-cell level. Fast-fatigable neurons (which are involved in movements like jumping and sprinting and are the first to die in ALS) were found to have the most MMP-9 protein, whereas slow neurons (which control posture and are only partially affected in ALS) had none. “So, MMP-9 is not only labeling the most vulnerable groups of motor neurons, it is labeling the most vulnerable subtypes within those groups, as well,” said Dr. Spiller.

In another experiment, the researchers tested whether MMP-9 has afunctional role in ALS by crossing MMP-9 knockout mice with SOD1 mutant mice (a standard mouse model of ALS). Progeny from this cross with no MMP-9 exhibited an 80-day delay in loss of fast-fatigable motor neuron function and a 25 percent longer lifespan, compared with littermates with two copies of the MMP-9 gene. “This effect on nerve-muscle synapses is the largest ever seen in a mouse model of ALS,” said Dr. Spiller.

The same effect on motor neuron function was seen when MMP-9 was inactivated in SOD1 mutant mice using chemical injections or virally mediated gene therapy.

“Even after treatment, these mice didn’t have a normal lifespan, so inactivating MMP-9 is not a cure,” said Dr. Henderson. “But it’s remarkable that lowering levels of a single gene could have such a strong effect on the disease. That’s encouraging for therapeutic purposes.”

The researchers are still investigating how MMP-9 affects motor neuron function. Their findings suggest that the protein plays a role in increasing stress on the endoplasmic reticulum, an organelle involved in transporting and processing materials within cells. “Our goal is to learn more about MMP-9 and related pathways and to identify a new set of therapeutic targets,” said Dr. Henderson.

Age no obstacle to nerve cell regeneration

In aging worms at least, it is insulin, not Father Time, that inhibits a motor neuron’s ability to repair itself — a finding that suggests declines in nervous system health may not be inevitable.

All organisms show a declining ability to regenerate damaged nervous systems with age, but the study appearing in the Feb. 5 issue of the journal Neuron suggests this deficit is not due to the ravages of time.

“The nervous system regulates its own response to age, separately from what happens in the rest of the body,” said Marc Hammarlund, assistant professor of genetics and senior author of the new study. “By manipulating the insulin pathway, we can make animals that live longer but have nervous systems that age normally, or conversely, we can make animals that die at a normal age but have a young nervous system.”

Alexandra Byrne, postdoctoral associate in genetics and lead author of the study, identified two genetic pathways that regulate insulin activity and are responsible for age-related declines in a worm’s ability to regenerate neuronal axons, or connective branches. The team pinpointed two other pathways that also regulate a neuron’s ability to regenerate, but that have no connection to the age of the worm.

The worm C. elegans is a well-established model to study the genetics of aging, and manipulation of the family of genes that regulate insulin activity has been shown to dramatically increase lifespan of the organism. The new study reveals that insulin signaling is also directly affecting the nervous system.

“We hope to understand how different pathways coordinately regulate neuronal aging, and more specifically, how to entice an aged neuron to regenerate after injury,” Byrne said.

“The hope is to increase healthspan, not just lifespan,” Hammarlund said.

Study provides new insights into cause of human neurodegenerative disease

A recent study led by scientists from the National University of Singapore (NUS) opens a possible new route for treatment of Spinal Muscular Atrophy (SMA), a devastating disease that is the most common genetic cause of infant death and also affects young adults. As there is currently no known cure for SMA, the new discovery gives a strong boost to the fight against SMA.

SMA is caused by deficiencies in the Survival Motor Neuron (SMN) gene. This gene controls the activity of various target genes. It has long been speculated that deregulation of some of these targets contributes to SMA, yet their identity remained unknown.

Using global genome analysis, the research team, led by Associate Professor Christoph Winkler of the Department of Biological Sciences at the NUS Faculty of Science and Dr Kelvin See, a former A*STAR graduate scholar in NUS who is currently a Research Fellow at the Genome Institute of Singapore (GIS), found that deficiency in the SMN gene impairs the function of the Neurexin2 gene. This in turn limits the neurotransmitter release required for the normal function of nerve cells. The degeneration of motor neurons in the spinal cord causes SMA. This is the first time that scientists establish an association between Neurexin2 and SMA.

Preliminary experimental data also showed that a restoration of Neurexin2 activity can partially recover neuron function in SMN deficient zebrafish. This indicates a possible new direction for therapy of neurodegeneration.

Collaborating with Assoc Prof Winkler and the NUS researchers are Dr S. Mathavan and his team at GIS, as well as researchers from the University of Wuerzburg in Germany. The breakthrough discovery was first published in scientific journal Human Molecular Genetics last month.

Small zebrafish provides insights into human neurodegenerative disease

SMA is a genetic disease that attacks a distinct type of nerve cells called motor neurons in the spinal cord. The disease has been found to be caused by a defect in the SMN gene, a widely used gene that is responsible for normal motor functions in the body.

To study how defects in SMN cause neuron degeneration, the scientists utilised a zebrafish model, as the small fish has a relatively simple nervous system that allows detailed imaging of neuron behaviour.

In laboratory experiments, the researchers showed when SMN activity in zebrafish was reduced to levels found in human SMA patients, Neurexin2 function was impaired. This novel disease mechanism was also discovered in other in vivo models, suggesting that it is applicable to mammals and possibly human patients.

When the scientists measured the activity of nerve cells in zebrafish using laser imaging, they found that nerve cells deficient for Neurexin2 or SMN could not be activated to the same level as healthy nerve cells. This impairment consequently led to the reduction of muscular activity. Interestingly, preliminary data showed that a restoration of Neurexin2 activity can partially recover neuron function in SMN deficient zebrafish.

Further studies

Assoc Prof Winkler, who is also with the NUS Centre for Biolmaging Sciences, explained, “These findings significantly advance our understanding of how the loss of SMN leads to neurodegeneration. A better understanding of these mechanisms will lead to novel therapeutic strategies that could aim at restoring and maintaining functions in deficient nerve cells of SMA patients.”

Dr See added, “Our study provides a link between SMN deficiency and its effects on a critical gene important for neuronal function. It would be interesting to perform follow up studies in clinical samples to further investigate the role of Neurexin2 in SMA pathophysiology.”

Moving forward, the team of scientists will conduct further research to determine if Neurexin2 is an exclusive mediator of SMN induced defects and hence can be used as a target for future drug designs. They hope their findings will contribute towards treatment of neurodegeneration.