Overlooked cells hold keys to brain organization and disease

Scientists studying brain diseases may need to look beyond nerve cells and start paying attention to the star-shaped cells known as “astrocytes,” because they play specialized roles in the development and maintenance of nerve circuits and may contribute to a wide range of disorders, according to a new study by UC San Francisco researchers.

In a study published online April 28, 2014 in Nature, the researchers report that malfunctioning astrocytes might contribute to neurodegenerative disorders such as Lou Gehrig’s disease (ALS), and perhaps even to developmental disorders such as autism and schizophrenia.

David Rowitch, MD, PhD, UCSF professor of pediatrics and neurosurgery and a Howard Hughes Medical Institute investigator, led the research.

The researchers discovered in mice that a particular form of astrocyte within the spinal cord secretes a protein needed for survival of the nerve circuitry that controls reflexive movements. This discovery is the first demonstration that different types of astrocytes exist to support development and survival of distinct nerve circuits at specific locations within the central nervous system.

Astrocytes vastly outnumber signal-conducting neurons, and make up the majority of cells in the brain. But where neuroscientists are accustomed to seeing only vanilla when it comes to astrocytes – viewing all of them as similar despite their different locations in brain and spinal cord — they now will have to imagine “31 flavors” or more.

There might even be hundreds of distinctive varieties of astrocytes performing specific functions in different locations, according to Rowitch, chief of neonatology for UCSF Benioff Children’s Hospital San Francisco.

"Our study shows roles for specialized astrocytes that function to support particular kinds of neurons in their neighborhood," Rowitch said.

Led by Rowitch lab postdoctoral fellow Anna Molofsky, MD, PhD, the researchers studied the spinal cord sensory motor circuit, which allows both mice and humans to react without thought – to jerk a limb away from something hot, for instance.

The team discovered that a protein called Sema3a is produced much more abundantly by astrocytes close to motor neurons than by astrocytes from other regions in the spinal cord. They concluded that motor neurons required this source of Sema3a from the local astrocytes, because when Sema3a production was blocked, the motor neurons failed to form normal connections, and half of them died.

Motor neurons also die in ALS, a fatal neurodegenerative disease, and in spinal muscular atrophy, a disease that can affect newborn infants. In other studies, scientists have found that abnormal astrocytes can have toxic effects on motor neurons.

Molofsky is a psychiatrist who studies how astrocytes organize nerve circuits, and how disruptions of these nerve circuits during development or disease may involve abnormal astrocyte function. Disrupted neural circuits are believed to be responsible for certain psychiatric disorders.

"The immediate implications of this study are for diseases of motor neurons, like ALS, but I think our findings might also apply more generally to diseases of neural-circuit formation in the brain such as autism, schizophrenia and epilepsy," Molofsky said. "To achieve a comprehensive understanding of how neural circuits form and are maintained, it seems important that we integrate knowledge of how astrocytes support that process."

Rowitch agrees. “To the extent that psychiatric or neurological disease is localized to a specific part of the brain, we should now be considering the potentially specialized type of astrocytes regulating nerve connections in that region and their contributions to disease,” he said.

(Image: Astrocytes surround neuronal sysnapses and form networks physically coupled by gap-junctions. Credit: Dr. Takahiro Takano)

(Image caption:The image depicts mice having a normal nerve (left) as compared to an incomplete nerve, a condition resulting in permanent downward gaze in both mice and humans. Image courtesy of Jeremy Duncan)

Researchers track down cause of eye mobility disorder

Imagine you cannot move your eyes up, and you cannot lift your upper eyelid. You walk through life with your head tilted upward so that your eyes look straight when they are rolled down in the eye socket. Obviously, such a condition should be corrected to allow people a normal position of their head. In order to correct this condition, one would need to understand why this happens.

In a paper published in the April 16 print issue of the journal Neuron, University of Iowa researchers Bernd Fritzsch and Jeremy Duncan and their colleagues at Harvard Medical School, along with investigator and corresponding author Elizabeth Engle, describe how their studies on mutated mice mimic human mutations.

It all started when Engle, a researcher at the Howard Hughes Medical Institute (HHMI), and Fritzsch, professor and departmental executive officer in the UI College of Liberal Arts and Sciences Department of Biology, began their interaction on the stimulation of eye muscles by their nerves, or “innervation,” around 20 years ago.

Approximately 10 years ago, Engle had identified the mutated genes in several patients with the eye movement disorder and subsequently developed a mouse with the same mutation she had identified in humans. However, while the effect on eye muscle innervation was comparable, there still was no clue as to why this should happen.

Fritzsch and his former biology doctoral student, Jeremy Duncan, worked with the Harvard researchers on a developmental study to find the point at which normal development of eye muscle innervations departs from the mutants. To their surprise, it happened very early in development. In fact, they found—only in mutant mice—a unique swelling in one of the nerves to the eye muscle.

More detailed analysis showed that these swellings came about because fibers extending to the eyes from the brain tried to leave the nerve as if they were already in the orbit, or eye socket. Since it happened so early, the researchers reasoned that something must be transported more effectively by this mutation to the motor neurons trying to reach the orbit and the eye muscles; something must be causing these motor neurons to assume they have already reached their target, the orbit of the eye.

To verify this enhanced function, the researchers developed another mouse that lacked the specific protein and found no defects in muscle innervation. Moreover, when they bred mice that carried malformed proteins with those that had none of these proteins, the mice developed a normal innervation.

This data provided clear evidence of what was going wrong and why, but it did not provide a clue as to the possible product that was more effectively transported in the mutant mice and, by logical extension, in humans. Further analysis revealed that breeding their mutant mice with another mutant having eye muscle innervation defects could enhance the effect of either mutation.

With this finding, they had identified the mutated protein, its enhanced function, and at least some of the likely cargo transported by this protein to allow normal innervation of eye muscles. This data provides the necessary level of understanding to design rational approaches to block the defect from developing.

Knowing what goes wrong and at what time during development can allow the problem to be corrected before it develops through proper manipulations. Engle, Fritzsch, and their collaborators currently are designing new approaches to rescue normal innervation in mice. In the future, their work may help families carrying such genetic mutations to have children with normal eye movement.

Muscle paralysis eased by light-sensitive stem cells

A genetic tweak can make light work of some nervous disorders. Using flashes of light to stimulate modified neurons can restore movement to paralysed muscles. A study demonstrating this, carried out in mice, lays the path for using such “optogenetic” approaches to treat nerve disorders ranging from spinal cord injury to epilepsy and motor neuron disease.

Optogenetics has been hailed as one of the most significant recent developments in neuroscience. It involves genetically modifying neurons so they produce a light-sensitive protein, which makes them “fire”, sending an electrical signal, when exposed to light.

So far optogenetics has mainly been used to explore how the brain works, but some groups are exploring using it as therapy. One stumbling block has been fears about irreversibly genetically manipulating the brain.

In the latest study, a team led by Linda Greensmith of University College London altered mouse stem cells in the lab before transplanting them into nerves in the leg – this means they would be easier to remove if something went wrong.

"It’s a very exciting approach that has a lot of potential," says Ziv Williams of Harvard Medical School in Boston.

Muscles in action

Greensmith’s team inserted an algal gene that codes for a light-responsive protein into mouse embryonic stem cells. They then added signalling molecules to make the stem cells develop into motor neurons, the cells that carry signals to and from the spinal cord to the rest of the body. They implanted these into the sciatic nerve – which runs from the spinal cord to the lower limbs – of mice whose original nerves had been cut.

After waiting five weeks for the implanted neurons to integrate with the muscle, Greensmith’s team anaesthetised the mice, cut open their skin and shone pulses of blue light on the nerve. The leg muscles contracted in response. “We were surprised at how well this worked,” says Greensmith.

Most current approaches being investigated to help people who are paralysed involve electrically stimulating their nerves or muscles. But this can be painful because they may still have working pain neurons. Plus, the electricity makes the muscles contract too forcefully, making them tire quickly.

Using the optogenetic approach, however, allows the muscle fibres to be stimulated more gently, because the light level can be increased with each pulse. “It gives a very smooth contraction,” says Greensmith.

Breathing restoration

To make the technique practical for use in people, the researchers are developing a light-emitting diode in the form of a cuff that would go around the nerve, which could be connected to a miniature battery pack under the skin.

They are also trying to develop an alternative to using embryonic stem cells, as these would require the recipient to take drugs to stop their immune system attacking the transplanted neurons. Instead the team is working with induced pluripotent stem cells, cells that have been reprogrammed to behave like embryonic stem cells, but can be made from a small sample of the intended recipient’s own skin.

The team’s first goal is to help people with motor neuron disease who lose the ability to control their breathing muscles. “Walking involves contracting about 40 different muscles in complex sequences,” says Greensmith. “Breathing is very simple – one muscle contracts and relaxes.”

They plan to test the restoration of breathing ability in pigs, and are developing a pacemaker that could repeatedly illuminate the phrenic nerve in the chest, which controls the diaphragm.

Other groups are exploring different therapeutic applications of optogenetics, including treatments for epilepsy and Parkinson’s disease

(Image caption: In this microscope photo of motor neurons created in the laboratory of Su-Chun Zhang, green marks the nucleus and red marks the nerve fibers. Zhang and co-workers at the Waisman Center have identified a misregulation of protein in the nucleus as the likely first step in the pathology of ALS. Credit: Hong Chen, Su-Chun Zhang/Waisman Center)

Study helps unravel the tangled origin of ALS

By studying nerve cells that originated in patients with a severe neurological disease, a University of Wisconsin-Madison researcher has pinpointed an error in protein formation that could be the root of amyotrophic lateral sclerosis.

Also called Lou Gehrig’s disease, ALS causes paralysis and death. According to the ALS Association, as many as 30,000 Americans are living with ALS.

After a genetic mutation was discovered in a small group of ALS patients, scientists transferred that gene to animals and began to search for drugs that might treat those animals. But that approach has yet to work, says Su-Chun Zhang, a neuroscientist at the Waisman Center at UW-Madison, who is senior author of the new report, published April 3 in the journal Cell Stem Cell.

Zhang has been using a different approach — studying diseased human cells in lab dishes. Those cells, called motor neurons, direct muscles to contract and are the site of failure in ALS.

About 10 years ago, Zhang was the first in the world to grow motor neurons from human embryonic stem cells. More recently, he updated that approach by transforming skin cells into iPS (induced pluripotent stem) cells that were transformed, in turn, into motor neurons.

IPS cells can be used as “disease models,” as they carry many of the same traits as their donor. Zhang says the iPS approach offers a key advantage over the genetic approach, which “can only study the results of a known disease-causing gene. With iPS, you can take a cell from any patient, and grow up motor neurons that have ALS. That offers a new way to look at the basic disease pathology.”

In the new report, Zhang, Waisman scientist Hong Chen, and colleagues have pointed a finger at proteins that build a transport structure inside the motor neurons. Called neurofilament, this structure moves chemicals and cellular subunits to the far reaches of the nerve cell. The cargo needing movement includes neurotransmitters, which signal the muscles, and mitochondria, which process energy.

Motor neurons that control foot muscles are about three feet long, so neurotransmitters must be moved a yard from their origin in the cell body to the location where they can signal the muscles, Zhang says. A patient lacking this connection becomes paralyzed; tellingly, the first sign of ALS is often paralysis in the feet and legs.

Scientists have known for some time that in ALS, “tangles” along the nerve’s projections, formed of misshapen protein, block the passage along the nerve fibers, eventually causing the nerve fiber to malfunction and die. The core of the new discovery is the source of these tangles: a shortage of one of the three proteins in the neurofilament.

The neurofilament combines structural and functional roles, Zhang says. “Like the studs, joists and rafters of a house, the neurofilament is the backbone of the cell, but it’s constantly changing. These proteins need to be shipped from the cell body, where they are produced, to the most distant part, and then be shipped back for recycling. If the proteins cannot form correctly and be transported easily, they form tangles that cause a cascade of problems.”

Finding neurofilament tangles in an autopsy of an ALS patient “will not tell you how they happen, when or why they happen,” Zhang says. But with millions of cells — all carrying the human disease — to work with, Zhang’s research group discovered the source of the tangles in the protein subunits that compose the neurofilaments. “Our discovery here is that the disease ALS is caused by misregulation of one step in the production of the neurofilament,” he says.

Beyond ALS, Zhang says “very similar tangles” appear in Alzheimer’s and Parkinson’s diseases. “We got really excited at the idea that when you study ALS, you may be looking at the root of many neurodegenerative disorders.”

While working with motor neurons sourced in stem cells from patients, Zhang says he and his colleagues saw “quite an amazing thing. The motor neurons we reprogrammed from patient skin cells were relatively young, and we found that the misregulation happens very early, which means it is the most likely cause of this disease. Nobody knew this before, but we think if you can target this early step in pathology, you can potentially rescue the nerve cell.”

In the experiment just reported, Zhang found a way to rescue the neural cells living in his lab dishes. When his group “edited” the gene that directs formation of the deficient protein, “suddenly the cells looked normal,” Zhang says.

Already, he reports, scientists at the Small Molecule Screening and Synthesis Facility at UW-Madison are looking for a way to rescue diseased motor neurons. These neurons are made by the millions from stem cells using techniques that Zhang has perfected over the years.

Zhang says “libraries” of candidate drugs, each containing a thousand or more compounds, are being tested. “This is exciting. We can put this into action right away. The basic research is now starting to pay off. With a disease like this, there is no time to waste.”

New hope for treating ALS

Patient stem cells help identify common problem, leading to clinical trials

Harvard stem cell scientists have discovered that a recently approved medication for epilepsy might be a meaningful treatment for amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease, a uniformly fatal neurodegenerative disorder. The researchers are now collaborating with Massachusetts General Hospital (MGH) to design an initial clinical trial testing the safety of the treatment in ALS patients.

The investigators all caution that a great deal of work needs to be done to assure the safety and efficacy of the treatment in ALS patients before physicians should start offering it.

The work, laid out in two related advance online publications in April by Cell Stem Cell and Cell Reports, is the long-term fruit of studies by Harvard Stem Cell Institute (HSCI) principal faculty member Kevin Eggan, who in a 2008 Science paper first raised the possibility of using ALS patient-derived stem cells to better understand the disease and identify therapeutic targets for new drugs.

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Team finds a better way to grow motor neurons from stem cells

Researchers report they can generate human motor neurons from stem cells much more quickly and efficiently than previous methods allowed. The finding, described in Nature Communications, will aid efforts to model human motor neuron development, and to understand and treat spinal cord injuries and motor neuron diseases such as amyotrophic lateral sclerosis (ALS).

The new method involves adding critical signaling molecules to precursor cells a few days earlier than previous methods specified. This increases the proportion of healthy motor neurons derived from stem cells (from 30 to 70 percent) and cuts in half the time required to do so.

“We would argue that whatever happens in the human body is going to be quite efficient, quite rapid,” said University of Illinois cell and developmental biology professor Fei Wang, who led the study with visiting scholar Qiuhao Qu and materials science and engineering professor Jianjun Cheng. “Previous approaches took 40 to 50 days, and then the efficiency was very low – 20 to 30 percent. So it’s unlikely that those methods recreate human motor neuron development.”

Qu’s method produced a much larger population of mature, functional motor neurons in 20 days.

The new approach will allow scientists to induce mature human motor neuron development in cell culture, and to identify the factors that are vital to that process, Wang said.

Stem cells are unique in that they can adopt the shape and function of a variety of cell types. Generating neurons from stem cells (either embryonic stem cells or those “induced” to revert back to an embryo-like state) requires adding signaling molecules to the cells at critical moments in their development.

Wang and other colleagues previously discovered a molecule (called compound C) that converts stem cells into “neural progenitor cells,” an early stage in the cells’ development into neurons. But further coaxing these cells to become motor neurons presented unusual challenges.

Previous studies added two important signaling molecules at Day 6 (six days after exposure to compound C), but with limited success in generating motor neurons. In the new study, Qu discovered that adding the signaling molecules at Day 3 worked much better: The neural progenitor cells quickly and efficiently differentiated into motor neurons.

This indicates that Day 3 represents a previously unrecognized neural progenitor cell stage, Wang said.

The new approach has immediate applications in the lab. Watching how stem cells (derived from ALS patients’ own skin cells, for example) develop into motor neurons will offer new insights into disease processes, and any method that improves the speed and efficiency of generating the motor neurons will aid scientists. The cells can also be used to screen for drugs to treat motor neuron diseases, and may one day be used therapeutically to restore lost function.

“To have a rapid, efficient way to generate motor neurons will undoubtedly be crucial to studying – and potentially also treating – spinal cord injuries and diseases like ALS,” Wang said.

Whole-Brain Activity Maps Reveal Stereotyped, Distributed Networks for Visuomotor Behavior

Most behaviors, even simple innate reflexes, are mediated by circuits of neurons spanning areas throughout the brain. However, in most cases, the distribution and dynamics of firing patterns of these neurons during behavior are not known. We imaged activity, with cellular resolution, throughout the whole brains of zebrafish performing the optokinetic response. We found a sparse, broadly distributed network that has an elaborate but ordered pattern, with a bilaterally symmetrical organization. Activity patterns fell into distinct clusters reflecting sensory and motor processing. By correlating neuronal responses with an array of sensory and motor variables, we find that the network can be clearly divided into distinct functional modules. Comparing aligned data from multiple fish, we find that the spatiotemporal activity dynamics and functional organization are highly stereotyped across individuals. These experiments systematically reveal the functional architecture of neural circuits underlying a sensorimotor behavior in a vertebrate brain.

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Researchers Use Computers to “See” Neurons to Better Understand Brain Function

A study conducted by local high school students and faculty from the Department of Computer and Information Science in the School of Science at Indiana University-Purdue University Indianapolis reveals new information about the motor circuits of the brain that may one day help those developing therapies to treat conditions such as stroke, schizophrenia, spinal cord injury or Alzheimer’s disease.

"MRI and CAT scans of the human brain can tell us many things about the structure of this most complicated of organs, formed of trillions of neurons and the synapses via which they communicate. But we are a long way away from having imaging techniques that can show single neurons in a complex brain like the human brain," said Gavriil Tsechpenakis, Ph.D., assistant professor of computer science in the School of Science at IUPUI.

"But using the tools of artificial intelligence, specifically computer vision and image processing, we are able to visualize and process actual neurons of model organisms. Our work in the brain of a model organism—the fruit fly—will help us and other researchers move forward to more complex organisms with the ultimate goal of reconstructing the human central nervous system to gain insight into what goes wrong at the cellular level when devastating disorders of the brain and spinal cord occur. This understanding may ultimately inform the treatment of these conditions," said Tsechpenakis.

In this study, which processed images and reconstructed neuronal motor circuitry in the brain, the researchers, who included two Indianapolis high school students—Rachel Stephens and Tiange (Tony) Qu—collected and analyzed data on minute structures over various developmental stages, efforts linking neuroscience and computer science.

"Both high school students who worked on this study performed neuroscience and computation efforts similar to that conducted elsewhere by graduate students. It was impressive to see what sophisticated and key work they could—with mentoring—do," said Tsechpenakis.

Qu said the work was initially rather scary and intimidating but that he rapidly grew to appreciate the opportunity to work in the School of Science lab. “Unlike high school, we were not told how to get from point A to point B. Dr. Tsechpenakis explained what point A and B were and taught us how to figure out how to get from A to B.” 

Qu, a 17-year-old senior at Ben Davis High School, now sees neuroscience as a potential college major with biomedical research as an eventual career goal. He continues to work in the lab after school focusing on change over time in fruit fly larvae motor neurons.

Stephens, a senior at North Central High School, said she enjoyed the collaborative nature of the research, with computer scientists and life scientists working together on a problem.

"Dr. Tsechpenakis made it clear to us that different perspectives are necessary, and the ability to think about a problem is more valuable than the education and training you’ve had,” she said. “Before I joined the lab I hadn’t really thought about how computer science could help heal." The 17-year-old plans a pre-med major in college and a career as a physician.

Experimental stroke drug also shows promise for people with Lou Gehrig’s disease

Keck School of Medicine of USC neuroscientists have unlocked a piece of the puzzle in the fight against Lou Gehrig’s disease, a debilitating neurological disorder that robs people of their motor skills. Their findings appear in the March 3, 2014, online edition of the Proceedings of the National Academy of Sciences of the United States of America, the official scientific journal of the U.S. National Academy of Sciences.

"We know that both people and transgenic rodents afflicted with this disease develop spontaneous breakdown of the blood-spinal cord barrier, but how these microscopic lesions affect the development of the disease has been unclear," said Berislav V. Zlokovic, M.D., Ph.D., the study’s principal investigator and director of the Zilkha Neurogenetic Institute at USC. "In this study, we show that early motor neuron dysfunction related to the disease in mice is proportional to the degree of damage to the blood-spinal cord barrier and that restoring the integrity of the barrier delays motor neuron degeneration. We are hopeful that we can apply these findings to the corresponding disease mechanism in people. "

In this study, Zlokovic and colleagues found that an experimental drug now being studied in human stroke patients appears to protect the blood-spinal cord barrier’s integrity in mice and delay motor neuron impairment and degeneration. The drug, an activated protein C analog called 3K3A-APC, was developed by Zlokovic’s start-up biotechnology company, ZZ Biotech.

Lou Gehrig’s disease, also called amyotrophic lateral sclerosis, or ALS, attacks motor neurons, which are cells that control the muscles. The progressive degeneration of the motor neurons in ALS eventually leads to paralysis and difficulty breathing, eating and swallowing.

According to The ALS Association, approximately 15 people in the United States are diagnosed with ALS every day. It is estimated that as many as 30,000 Americans live with the disease. Most people who develop ALS are between the ages of 40 and 70, with an average age of 55 upon diagnosis. Life expectancy of an ALS patient averages about two to five years from the onset of symptoms.

ALS’s causes are not completely understood, and no cure has yet been found. Only one Food and Drug Administration-approved drug called riluzole has been shown to prolong life by two to three months. There are, however, devices and therapies that can manage the symptoms of the disease to help people maintain as much independence as possible and prolong survival.