ALS progression linked to increased protein instability

The new study provides evidence that proteins linked to more severe forms of ALS are less stable structurally and more prone to form clusters or aggregates. Mutants of the superoxide dismutase (SOD) protein formed long, rod-shaped aggregates (shown here as red lattice), compared to the compact folded structure of wild-type SOD (purple ribbons). Credit: Image courtesy of the Getzoff and Tainer labs, The Scripps Research Institute.

A new study by scientists from The Scripps Research Institute (TSRI), Lawrence Berkeley National Laboratory (Berkeley Lab) and other institutions suggests a cause of amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease.

"Our work supports a common theme whereby loss of protein stability leads to disease," said John A. Tainer, professor of structural biology at TSRI and senior scientist at Berkeley Lab, who shared senior authorship of the new research with TSRI Professor Elizabeth Getzoff.

Getzoff, Tainer and their colleagues, who focused on the effects of mutations to a gene coding for a protein called superoxide dismutase (SOD), report their findings this week in the online Early Edition of the Proceedings of the National Academy of Sciences. The study provides evidence that those proteins linked to more severe forms of the disease are less stable structurally and more prone to form clusters or aggregates.

"The suggestion here is that strategies for stabilizing SOD proteins could be useful in treating or preventing SOD-linked ALS," said Getzoff.

Striking in the Prime of Life

ALS is notorious for its ability to strike down people in the prime of life. It first leapt into public consciousness when it afflicted baseball star Lou Gehrig, who succumbed to the disease in 1941 at the age of only 38. Recently, the ALS Association's Ice Bucket Challenge has enhanced public awareness of the disease.

ALS kills by destroying muscle-controlling neurons, ultimately including those that control breathing. At any one time, about 10,000 Americans are living with the disease, according to new data from the Centers for Disease Control and Prevention, but it is almost always lethal within several years of the onset of symptoms.

SOD1 mutations, the most studied factors in ALS, are found in about a quarter of hereditary ALS cases and seven percent of ordinary "sporadic" ALS cases. SOD-linked ALS has nearly 200 variants, each associated with a distinct SOD1 mutation. Scientists still don't agree, though, on just how the dozens of different SOD1 mutations all lead to the same disease.

One feature that SOD1-linked forms of ALS do have in common is the appearance of SOD clusters or aggregates in affected motor neurons and their support cells. Aggregates of SOD with other proteins are also found in affected cells, even in ALS cases that are not linked to SOD1 mutations.

In 2003, based on their and others' studies of mutant SOD proteins, Tainer, Getzoff and their colleagues proposed the "framework destabilization" hypothesis. In this view, ALS-linked mutant SOD1 genes all code for structurally unstable forms of the SOD protein. 
Inevitably some of these unstable SOD proteins lose their normal folding enough to expose sticky elements that are normally kept hidden, and they begin to aggregate with one another, faster than neuronal cleanup systems can keep up -- and that accumulating SOD aggregation somehow triggers disease.

Faster Clumping, Worse Disease

In the new study, the Tainer and Getzoff laboratories and their collaborators used advanced biophysical methods to probe how different SOD1 gene mutations in a particular genetic ALS "hotspot" affect SOD protein stability.

To start, they examined how the aggregation dynamics of the best-studied mutant form of SOD, known as SOD G93A, differed from that of non-mutant, "wild-type" SOD. To do this, they developed a method for gradually inducing SOD aggregation, which was measured with an innovative structural imaging system called SAXS (small-angle X-ray scattering) at Berkeley Lab's SIBYLS beamline.

"We could detect differences between the two proteins even before we accelerated the aggregation process," said David S. Shin, a research scientist in Tainer's laboratories at Berkeley Lab and TSRI who continues structural work on SOD at Berkeley.

The G93A SOD aggregated more quickly than wild-type SOD, but more slowly than an SOD mutant called A4V that is associated with a more rapidly progressing form of ALS.

Subsequent experiments with G93A and five other G93 mutants (in which the amino acid glycine at position 93 on the protein is replaced with a different amino acid) revealed that the mutants formed long, rod-shaped aggregates, compared to the compact folded structure of wild-type SOD. The mutant SOD proteins that more quickly formed longer aggregates were again those that corresponded to more rapidly progressing forms of ALS.

What could explain these SOD mutants' diminished stability? Further tests focused on the role of a copper ion that is normally incorporated within the SOD structure and helps stabilize the protein. Using two other techniques, electron-spin resonance (ESR) spectroscopy and inductively coupled plasma mass spectrometry (ICP-MS), the researchers found that the G93-mutant SODs seemed normal in their ability to take up copper ions, but had a reduced ability to retain copper under mildly stressing conditions -- and this ability was lower for the SOD mutants associated with more severe ALS.

"There were indications that the mutant SODs are more flexible than wild-type SOD, and we think that explains their relative inability to retain the copper ions," said Ashley J. Pratt, the first author of the study, who was a student in the Getzoff laboratory and postdoctoral fellow with Tainer at Berkeley Lab.

Toward New Therapies

In short, the G93-mutant SODs appear to have looser, floppier structures that are more likely to drop their copper ions -- and thus are more likely to misfold and stick together in aggregates.

Along with other researchers in the field, Getzoff and Tainer suspect that deviant interactions of mutant SOD trigger inflammation and disrupt ordinary protein trafficking and disposal systems, stressing and ultimately killing affected neurons.

"Because mutant SODs get bent out of shape more easily," said Getzoff, "they don't hold and release their protein partners properly. By defining these defective partnerships, we can provide new targets for the development of drugs to treat ALS."

The researchers also plan to confirm the relationship between structural stability and ALS severity in other SOD mutants.

"If our hypothesis is correct," said Shin, "future therapies to treat SOD-linked ALS need not be tailored to each individual mutation -- they should be applicable to all of them."

Source: The Scripps Research Institute

Heart drug may help treat ALS, mouse study shows

In the top image, cells from a mouse model of amyotrophic lateral sclerosis caused normal healthy brain cells (green) to die. But when scientists blocked an enzyme in the cells from the mouse model, more of the normal cells and their branches survived (bottom). Credit: Nature Neuroscience

Digoxin, a medication used in the treatment of heart failure, may be adaptable for the treatment of amyotrophic lateral sclerosis (ALS), a progressive, paralyzing disease, suggests new research at Washington University School of Medicine in St. Louis.

ALS, also known as Lou Gehrig's disease, destroys the nerve cells that control muscles. This leads to loss of mobility, difficulty breathing and swallowing and eventually death. Riluzole, the sole medication approved to treat the disease, has only marginal benefits in patients.

But in a new study conducted in cell cultures and in mice, scientists showed that when they reduced the activity of an enzyme or limited cells' ability to make copies of the enzyme, the disease's destruction of nerve cells stopped. The enzyme maintains the proper balance of sodium and potassium in cells.

"We blocked the enzyme with digoxin," said senior author Azad Bonni, MD, PhD. "This had a very strong effect, preventing the death of nerve cells that are normally killed in a cell culture model of ALS."

The findings appear online Oct. 26 in Nature Neuroscience.

The results stemmed from Bonni's studies of brain cells' stress responses in a mouse model of ALS. The mice have a mutated version of a gene that causes an inherited form of the disease and develop many of the same symptoms seen in humans with ALS, including paralysis and death.

Efforts to monitor the activity of a stress response protein in the mice unexpectedly led the scientists to another protein: sodium-potassium ATPase. This enzyme ejects charged sodium particles from cells and takes in charged potassium particles, allowing cells to maintain an electrical charge across their outer membranes.

Maintenance of this charge is essential for the normal function of cells. The particular sodium-potassium ATPase highlighted by Bonni's studies is found in nervous system cells called astrocytes. In the ALS mice, levels of the enzyme are higher than normal in astrocytes.

Bonni's group found that the increase in sodium-potassium ATPase led the astrocytes to release harmful factors called inflammatory cytokines, which may kill motor neurons.

Recent studies have suggested that astrocytes may be crucial contributors to neurodegenerative disorders such as ALS, and Alzheimer's, Huntington's and Parkinson's diseases. For example, placing astrocytes from ALS mice in culture dishes with healthy motor neurons causes the neurons to degenerate and die.

"Even though the neurons are normal, there's something going on in the astrocytes that is harming the neurons," said Bonni, the Edison Professor of Neurobiology and head of the Department of Anatomy and Neurobiology.

How this happens isn't clear, but Bonni's results suggest the sodium-potassium ATPase plays a key role. When he conducted the same experiment but blocked the enzyme in ALS astrocytes using digoxin, the normal motor nerve cells survived. Digoxin blocks the ability of sodium-potassium ATPase to eject sodium and bring in potassium.

In mice with the mutation for inherited ALS, those with only one copy of the gene for sodium-potassium ATPase survived an average of 20 days longer than those with two copies of the gene. When one copy of the gene is gone, cells make less of the enzyme.

"The mice with only one copy of the sodium-potassium ATPase gene live longer and are more mobile," Bonni said. "They're not normal, but they can walk around and have more motor neurons in their spinal cords."

Many important questions remain about whether and how inhibitors of the sodium-potassium ATPase enzyme might be used to slow progressive paralysis in ALS, but Bonni said the findings offer an exciting starting point for further studies.

Source: Washington University School of Medicine

Existing drug, riluzole, may prevent foggy 'old age' brain, research shows

Better memory makers: When researchers looked at certain neurons (similar to the one shown on top) in rats treated with riluzole, they found an important change in one brain region, the hippocampus: more clusters of so-called spines, receiving connections that extend from the branches of a neuron (bottom). Credit: Image courtesy of Rockefeller University

Forgetfulness, it turns out, is all in the head. Scientists have shown that fading memory and clouding judgment, the type that comes with advancing age, show up as lost and altered connections between neurons in the brain. But new experiments suggest an existing drug, known as riluzole and already on the market as a treatment for ALS, may help prevent these changes.

Researchers at The Rockefeller University and The Icahn School of Medicine at Mount Sinai found they could stop normal, age-related memory loss in rats by treating them with riluzole. This treatment, they found, prompted changes known to improve connections, and as a result, communication, between certain neurons within the brain's hippocampus.

"By examining the neurological changes that occurred after riluzole treatment, we discovered one way in which the brain's ability to reorganize itself -- its neuroplasticity -- can be marshaled to protect it against some of the deterioration that can accompany old age, at least in rodents," says co-senior study author Alfred E. Mirsky Professor Bruce McEwen, head of the Harold and Margaret Milliken Hatch Laboratory of Neuroendocrinology. The research is published this week in Proceedings of the National Academy of Sciences.

Neurons connect to one another to form circuits connecting certain parts of the brain, and they communicate using a chemical signal known as glutamate. But too much glutamate can cause damage; excess can spill out and excite connecting neurons in the wrong spot. In the case of age-related cognitive decline, this process damages neurons at the points where they connect -- their synapses. In neurodegenerative disorders, such as Alzheimer's disease, this contributes to the death of neurons.

Used to slow the progress of another neurodegenerative condition, ALS (also known as Lou Gehrig's disease), riluzole was an obvious choice as a potential treatment, because it works by helping to control glutamate release and uptake, preventing harmful spillover. The researchers began giving riluzole to rats once they reached 10 months old, the rat equivalent of middle age, when their cognitive decline typically begins.

After 17 weeks of treatment, the researchers tested the rats' spatial memory -- the type of memory most readily studied in animals -- and found they performed better than their untreated peers, and almost as well as young rats. For instance, when placed in a maze they had already explored, the treated rats recognized an unfamiliar arm as such and spent more time investigating it.

When the researchers looked inside the brains of riluzole-treated rats, they found telling changes to the vulnerable glutamate sensing circuitry within the hippocampus, a brain region implicated in memory and emotion.

"We have found that in many cases, aging involves synaptic changes that decrease synaptic strength, the plasticity of synapses, or both," said John Morrison, professor of neuroscience and the Friedman Brain Institute and dean of basic sciences and the Graduate School of Biomedical Sciences at Mount Sinai. "The fact that riluzole increased the clustering of only the thin, most plastic spines, suggests that its enhancement of memory results from both an increase in synaptic strength and synaptic plasticity, which might explain its therapeutic effectiveness."

In this case, the clusters involved thin spines, a rapidly adaptable type of spine. The riluzole-treated animals had more clustering than the young animals and their untreated peers, who had the least. This discovery led the researchers to speculate that, in general, the aged brain may compensate by increasing clustering. Riluzole appears to enhance this mechanism.

"In our study, this phenomenon of clustering proved to be the core underlying mechanism that prevented age-related cognitive decline. By compensating the deleterious changes in glutamate levels with aging and Alzheimer's disease and promoting important neuroplastic changes in the brain, such as clustering of spines, riluzole may prevent cognitive decline," says first author Ana Pereira, an instructor in clinical investigation in McEwen's laboratory.

Taking advantage of the overlap of neural circuits vulnerable to age-related cognitive decline and Alzheimer's disease, Pereira is currently conducting a clinical trial to test the effectiveness of riluzole for patients with mild Alzheimer's.

Source: Rockefeller University

Ultrasound guides tongue to pronounce 'R' sounds

Using ultrasound technology to visualize the tongue's shape and movement can help children with difficulty pronouncing "r" sounds, according to research led by NYU Steinhardt assistant professor Tara McAllister Byun. Credit: Ramsay de Give / NYU Steinhardt

Using ultrasound technology to visualize the tongue's shape and movement can help children with difficulty pronouncing "r" sounds, according to a small study by NYU's Steinhardt School of Culture, Education, and Human Development and Montclair State University.

The ultrasound intervention was effective when individuals were allowed to make different shapes with their tongue in order to produce the "r" sound, rather than being instructed to make a specific shape. The findings appear online in the Journal of Speech, Language, and Hearing Research.

The "r" sound is one of the most frequent speech errors, and can be challenging to correct. For other sounds -- such as "t" or "p" -- speech pathologists can give clear verbal, visual or tactile cues to help children understand how the sound is created. "R" is difficult to show or describe in an easy-to-understand fashion.

In addition, most speech sounds are produced in the same way, but with "r," normal speakers use widely different tongue shapes to create the sound. The two primary strategies to create the "r" sound include a retroflex tongue shape, where the tongue tip is pointed up, and the bunched tongue shape, where the tongue tip is pointed down and body of tongue bunches up toward the top of the mouth.

Up to 10 percent of children have speech sound disorders, according to the National Institutes of Health. Some children respond well to conventional forms of speech therapy, but others have errors that persist despite their therapists' best efforts. A growing body of evidence suggests that treatment incorporating visual biofeedback, which uses various technologies to create a dynamic visual representation of speech, could fill this need.

"The idea that you could get around the challenges with 'r' sounds by showing children their tongues as they are talking is really appealing to clinicians," says Tara McAllister Byun, an assistant professor in NYU Steinhardt's Department of Communicative Sciences and Disorders and the study's lead author. "That's what ultrasound technology lets us do."

Linguists have used ultrasound in the past to study basic functions of speech, and in recent years, speech pathologists have begun exploring using ultrasound to treat children with speech errors. An ultrasound probe -- similar to ones used in cardiac and tissue imaging -- is held under the chin, and sound waves capture real-time images of the tongue. The images provide both the child and speech pathologist with information about the tongue's position and shape.

Using the ultrasound images as a guide, children learn how to manipulate their tongues, and speech pathologists advise them on how to make adjustments to better achieve different sounds.

Several case studies and small studies suggest that ultrasound biofeedback can successfully correct "r" speech errors. Byun and her colleagues set out to gather systematic evidence on the effectiveness of the treatment, studying eight children with difficulty pronouncing "r" sounds. Seven of the eight had previous speech therapy that was unsuccessful.

Four children participated in the initial eight-week study. They were taught to make a bunched tongue shape, guided by ultrasound, in an effort to better pronounce "r." The researchers saw only small improvements among the four participants.

However, while trying to create a bunched tongue, one child stumbled upon a retroflex tongue shape and was able to improve her "r" sound. As a result of her success, the researchers altered their study design to allow participants to choose their own tongue shape, with individualized guidance from speech language pathologists.

A different four children participated in the second study over an eight-week period. Using ultrasound to visualize their tongues, all four participants in the second study showed significant improvement in their "r" sounds.

"Our second study offers evidence that when flexibility is given to choose a tongue shape, rather than a one-size-fits all approach, ultrasound biofeedback treatment can be a highly effective intervention for children with trouble pronouncing 'r' sounds," Byun says.

The researchers noted that the two studies were not a controlled comparison, thus additional systematic research is needed before drawing strong conclusions about the importance of individualized tongue shapes.

Source: New York University