In a breakthrough study to be published in the May 23 issue of the Proceedings of the National Academy of Sciences, scientists at Northwestern University report new insights into the biological and molecular underpinnings of an entire class of neurodegenerative diseases, ranging from Huntington’s and Alzheimer’s diseases to cystic fibrosis and Creutzfeldt-Jakob disease, the human form of mad cow disease.
The findings provide a new model for understanding the common pathology of these diseases, knowledge that could lead to the development of effective drugs.
The diseases, characterized by the loss of nerve function, are caused by a variety of mutant genes. However, they all have one element in common: misfolded proteins that lead to protein aggregation, the accumulation of insoluble proteins that can result in toxicity and disease.
In their study, Richard I. Morimoto, John Evans Professor of Biology, and his research team zeroed in on this commonality, focusing on the dangerous protein behavior itself instead of the genes responsible. They discovered that polyglutamine aggregates (one type of protein aggregate) are indeed toxic and that these unhealthy proteins bring healthy and otherwise normal proteins to aggregate with them. The researchers also found that the growth of these aggregates can be suppressed by molecular chaperones called heat shock proteins.
“This work highlights how basic science can answer important questions in terms of human neurodegenerative diseases,” said Harry Orr, professor and director of the Institute of Human Genetics at the University of Minnesota Medical School. “Humans share much of the same biology with the subject of the study, the worm called C. elegans. The question that’s being asked, whether a neuron lives or dies, is a fundamental one across species.”
Proteins, made up of different combinations of amino acids, are basic components of all living cells. To do their job properly, each protein first must fold itself into the proper shape. In this delicate process, the protein receives its folding instructions from its amino acid sequence and is assisted by a class of proteins known as heat shock proteins or molecular chaperones that function to prevent misfolding, or, in the case of already misfolded proteins, to detect them and prevent their further accumulation.
“We believe that in neurodegenerative diseases there is a race between the body’s emergency crew of heat shock proteins and the protein misfolding that drives the formation of dangerous aggregates in the cell,” said Morimoto, who was first to clone a human heat shock gene in 1985. “When a more critical distress call arises in another part of the body -in the heart, for example -the heat shock proteins are diverted, giving the misfolded proteins a chance to develop into a protein aggregate. If the aggregate grows large enough, disease sets in and protein misfolding wins.”
In Huntington’s disease, for example, the mutated gene codes for a protein that produces an increasing number of consecutive residues of the amino acid glutamine. When the number of residues expands past 40, the protein becomes insoluble, causing the protein to misfold. This results in a loss of function and protein aggregation -in other words, disease. Humans without the mutated gene also may have an expansion of glutamine residues, but they remain healthy and unaffected because the number of residues in their cells never passes the dangerous 40 mark.
“Our model is the first to visually show the general principles underlying the behavior of polyglutamine expansions in cells,” said Morimoto. “These findings help us better understand the progressive nature of neurodegenerative diseases, particularly Huntington’s.”
In order to visualize misfolded proteins and their behavior in an animal, Morimoto and his team studied polyglutamine expansions expressed in C. elegans, a transparent roundworm whose genome, or complete genetic sequence, is known.
The researchers injected one group of healthy worms with cells with 19 glutamine residues and another group with cells with 82 glutamine residues. The glutamine residues were attached to a fluorescent reporter, allowing the researchers to view the glutamine residues, protein misfolding and any aggregation in the body wall muscle cells of the worms.
The worms with the soluble protein containing 19 glutamine residues exhibited healthy behavior, moving with normal speed and growing from embryo to adulthood in the expected time of 48 hours. On the other hand, the worms with 82 residues were extremely sluggish, and their growth was slowed. It took them more than twice as long to reach adulthood. Under a microscope, the researchers could see that the fluorescent green glutamine residues were distributed evenly throughout the muscle in the healthy worms, but the residues clearly were aggregated and lumped together in the unhealthy worms. The chronically stressed cells of the unhealthy worms activated the heat shock response.
Morimoto and his team wanted to see if the presence of molecular chaperones had any impact on the aggregates. The researchers co-expressed different yeast heat shock proteins in the worms with the 82 residues. They found that heat shock protein 104 reduced the appearance of polyglutamine aggregates and the associated developmental delay. Other molecular heat shock proteins had no effect. This key finding could aid the development of therapeutic strategies to combat the negative effects of the polyglutamine expansions.
“This is where the race between the body’s heat shock proteins and protein misfolding comes in,” said Morimoto. “Environmental and physiological stress intensifies misfolding events throughout the body and can divert the molecular chaperones away from the aggregates, accelerating the onset of disease.”
The researchers next asked whether or not the soluble and healthy protein with 19 residues would be affected by the insoluble and unhealthy protein with 82 residues. By co-expressing these two proteins in C. elegans, they discovered that the healthy protein co-aggregated in a pattern similar to that of the unhealthy protein alone. This indicates that the aggregation has a compounding effect, taking good proteins down with the bad.
“Rick Morimoto has developed a novel experimental model that promises to help us understand how protein aggregates form and why they are toxic to cells,” said Arthur Horwich, professor of genetics and pediatrics at Yale University and a Howard Hughes Medical Institute investigator. “He and his team have demonstrated the disabling effects of aggregation and at the same time have shown that molecular chaperones can alleviate some of these effects. It’s very encouraging work.”