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

Infectious prion protein discovered in urine of patients with variant Creutzfeldt-Jakob disease

Claudio Soto, Ph.D., in one of his labs at The University of Texas Health Science Center at Houston (UTHealth). Credit: Image courtesy of University of Texas Health Science Center at Houston

The misfolded and infectious prion protein that is a marker for variant Creutzfeldt-Jakob disease – linked to the consumption of infected cattle meat – has been detected in the urine of patients with the disease by researchers at The University of Texas Health Science Center at Houston (UTHealth) Medical School.

The results of the international study, led by Claudio Soto, Ph.D., professor of neurology at the UTHealth Medical School, will be published in the Aug. 7 issue of the New England Journal of Medicine.

Variant Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy in animals – also known as Mad Cow disease – are fatal neurodegenerative disorders. There are currently no noninvasive tools available to diagnose the disease and there are no treatments.

Sporadic Creutzfeldt-Jakob disease occurs worldwide at a rate of around 1 new case per million people per year. The variant form is a new disease occurring in people who either ate the beef of cows with bovine spongiform encephalopathy or, in the case of three patients in the United Kingdom, received blood transfusions from asymptomatic infected donors.

The international team of researchers analyzed urine samples from 68 patients with sporadic Creutzfeldt-Jakob disease, 14 patients with variant Creutzfeldt-Jakob disease, four patients with genetic prion diseases, 50 patients with other neurodegenerative diseases, 50 patients with nondegenerative neurologic diseases and 52 healthy persons.

Soto’s laboratory used a protein misfolding cyclic amplification assay, invented in the lab, which mimics the prion replication process in vitro that occurs in prion disease. The misfolded prion proteins were detected in the urine of 13 of 14 patients with variant Creutzfeldt-Jakob disease. The single patient whose urine was negative had been receiving an experimental treatment of pentosan polysulfate directly into the brain. No misfolded prion proteins were detected in the urine of any the other study subjects, including the patients who had sporadic Creutzfeldt-Jakob disease.

“What could be less invasive than detecting this disease in urine? The fact that we were able to detect just the variant Creutzfeldt-Jakob disease form in the urine is very important. This could lead to the development of commercial technology for diagnosis as well as to determine the safety of donated blood and urinary products,” said Soto, who is the director of The George and Cynthia Mitchell Center for Research in Alzheimer’s disease and Related Brain Disorders, and founder of Amprion Inc, a biotech company developing the cyclic amplification technology for commercial application.

According to the World Health Organization (WHO), variant Creutzfeldt-Jakob disease affects younger patients, who have a median age of 28 at death, compared to sporadic Creutzfeldt-Jakob disease with a median age of 68. Most patients, after diagnosis of either form, live less than a year before death.

As of June 2, 2014, 177 of 229 people in the world with Creutzfeldt-Jakob disease were from the United Kingdom. A 2013 study published in the British Medical Journal has estimated that approximately 30,000 people in the United Kingdom might be carriers of the variant form of the disease.

“This study reports, for the first time, the detection of the abnormal prion protein in the urine from patients with variant Creutzfeldt-Jakob disease using the protein misfolding amplification technique pioneered by Dr. Claudio Soto,” said co-author James W. Ironside, FMedSci, FRSE, professor of clinical neuropathology at the National CJD Research and Surveillance Unit at the University of Edinburgh. “This has great potential to allow the development of a highly sensitive and specific non-invasive test that can be used for the diagnosis of variant Creutzfeldt-Jakob disease, and potentially as a screening tool for variant Creutzfeldt-Jakob disease infection in asymptomatic individuals, which is a topic of current interest in the United Kingdom.”

Source: University of Texas Health Science Center at Houston

What bank voles can teach us about prion disease transmission and neurodegeneration

This image shows accumulation of misfolded, toxic prion protein (brown staining) in the brain of a transgenic mouse expressing bank vole PrP and challenged with human variant Creutzfeldt-Jakob disease (vCJD) prions. Credit: Image courtesy of Dr. Joel Watts

When cannibals ate brains of people who died from prion disease, many of them fell ill with the fatal neurodegenerative disease as well. Likewise, when cows were fed protein contaminated with bovine prions, many of them developed mad cow disease. On the other hand, transmission of prions between species, for example from cows, sheep, or deer to humans, is -- fortunately -- inefficient, and only a small proportion of exposed recipients become sick within their lifetimes.

A study published on April 3rd in PLOS Pathogens takes a close look at one exception to this rule: bank voles appear to lack a species barrier for prion transmission, and their universal susceptibility turns out to be both informative and useful for the development of strategies to prevent prion transmission.
Prions are misfolded, toxic versions of a protein called PrP, which in its normal form is present in all mammalian species that have been examined. Toxic prions are "infectious"; they can induce existing, properly folded PrP proteins to convert into the disease-associated prion form. Prion diseases are rare, but they share features with more common neurodegenerative diseases like Alzheimer's disease.

Trying to understand the unusual susceptibility of bank voles to prions from other species, Stanley Prusiner, Joel Watts, Kurt Giles and colleagues, from the University of California in San Francisco, USA, first tested whether the susceptibility is an intrinsic property of the voles' PrP, or whether other factors present in these rodents make them vulnerable.

The scientists introduced into mice the gene that codes for the normal bank vole prion protein, thereby generating mice that express bank vole PrP, but not mouse PrP. When these mice get older, some of them spontaneously develop neurologic illness, but in the younger ones the bank vole PrP is in its normal, benign folded state. The scientists then exposed young mice to toxic misfolded prions from 8 different species, including human, cattle, elk, sheep, and hamster.

They found that all of these foreign-species prions can cause prion disease in the transgenic mice, and that the disease develops often more rapidly than it does in bank voles. The latter is likely because the transgenic mice express higher levels of bank vole PrP than are naturally present in the voles.

The results show that the universal susceptibility of bank voles to cross-species prion transmission is an intrinsic property of bank vole PrP. Because the transgenic mice develop prion disease rapidly, the scientists propose that the mice will be useful tools in studying the processes by which toxic prions "convert" healthy PrP and thereby destroy the brain. And because that process is similar across many neurodegenerative diseases, better understanding prion disease development might have broader implications.

Source:  PLOS