Breakthrough Study of Toxic Protein 'Clumps' Shines Light on ALS

One of the most baffling diseases to researchers and doctors alike is amyotrophic lateral sclerosis (ALS). Also known as Lou Gehrig’s disease, it's a fatal, neurodegenerative condition that kills motor neurons, and as of yet has eluded the discovery of a cure. Up to 30,000 people in the U.S. are affected, and as many as 5000 new cases develop every year. Over time, patients with ALS become paralyzed, unable to move, speak, swallow, and eventually breathe, though their mental faculties remain mostly untouched. British physicist Stephen Hawking is one of the most well-known patients to live with the condition.

However, researchers at the University of North Carolina (UNC) School of Medicine recently published a study in Proceedings of the National Academy of Sciences that shows how a protein called SOD1 forms aggregates or “clumps” that kill motor neuron cells grown in the laboratory. This breakthrough study is the first to provide confirmation that the SOD1 proteins, found in the spinal cords of ALS patients, are toxic to their motor neurons—and offers hope for a drug cure.

“ALS is not one disease—it’s many diseases that have common symptoms,” says Nikolay Dokholyan, professor of biochemistry and biophysics at UNC, whose lab conducted the study. Patients are diagnosed with ALS when they lose muscle function due to motor neuron loss.

PROTEIN AGGREGATIONS ARE COMMON TO NEURODEGENERATIVE DISEASES

Dokholyan has been studying the SOD1 protein specifically for 14 years. “Over the years researchers have identified many ways for motor neurons to die. ALS is multi-factorial. We have been focusing on SOD1 to understand why this protein aggregates, and what triggers aggregation,” he tells mental_floss. “A lot of neurodegenerative diseases have things in common, like protein aggregation, such as Alzheimer’s and Parkinson’s. It’s not clear if aggregation itself is responsible for cell death or if it is actually protective of something.”

Many of these aggregated clumps disintegrate so rapidly it has been difficult to study them. “We knew the SOD1 protein is key to toxicity, but then we were after the biggest challenge of my life—to understand its structure,” Dokholyan says.

The study of SOD1 was further complicated by the way some of the molecules “hide” within the clump. Dokholyan compares the proteins to beads on a string. “Because of laws of attraction, they form these globules," he says. "Some of the beads will be inside the spherical molecule, and some on the surface.”

USING DIGESTIVE ENZYMES TO "CHOP" THE PROTEINS 

Because of this, it was difficult to determine whether the clumps were trimers or tetramers—three or four molecules. “By changing certain conditions of the surroundings of the trimer, we saw it fall apart before our eyes into single monomers," he says. "It was really beautiful."

Dokholyan and his team used high-speed atomic force microscopy (HS-AFM) to study the proteins' structures and make metric measurements of their volume. From there, they used a process called proteolysis, in which digestive enzymes called proteases were used to “chop” the proteins. “We would quench the process as soon as the proteases made the first cut,” he says. “The proteases were likely to cut the surface of the protein, but not inside.” Through this process, they could confirm the SOD1 structure to be trimers, comprised of only three molecules. 

Dokholyan credits Elizabeth Proctor, a graduate student in his lab at the time, and the study’s first author, with creating the computer algorithm that mapped all the experimental information to build a comprehensive structure of the proteins. “Nobody would believe a computer generation, so we had to prove that what we found is real,” he says.

Proctor then created several mutations of SOD1 structure—those that were more stabilized and formed more trimers, and those that destabilized, and formed fewer trimers. “Then we went to the lab and tested those mutants,” he says. They put these mutant proteins into living motor neuron cells that had been hybridized with cancer cells so that they would divide, since motor neurons don’t divide on their own.

“Every cell forming more trimers were killing cells,” Dokholyan says. “That told us that trimers are toxic. Increasing the population of trimers correlated with cell viability.”

NEXT STEP: ANALYZE THE GLUE 

This research opens doors to test drugs that could potentially stop the formation of these trimers. “In the ALS field there is only one drug approved. It prolongs life by a couple months but doesn’t stop progression. The key to finding a cure for ALS is understanding how proteins produced by the disease are behaving or misbehaving,” Dokholyan says. 

Since other neurodegenerative diseases like Alzheimer’s and Parkinson’s see problems with protein aggregates (amyloid in the former, tau in the latter) the information about SOD1 trimers’ toxicity may help understand the ways the other proteins affect other neurons.

While Dokholyan’s study is revelatory, more study is necessary to understand just how and why these aggregates cause cell death. “The more we know about how it progresses at the cellular and molecular level, the more chances we have to interfere with actual players that kill cells,” he says.

Dokholyan is excited for the next step of research, which will look at the “glue” that holds the trimers together, and which may shed light on other neurodegenerative diseases.