Neuronal cells clogged with a mutant protein associated with amyotrophic lateral sclerosis (ALS) die within hours after the clumps first form, researchers report in this month's
"This is the first study that has shown reasonable evidence that aggregates are involved in neurotoxicity," said neurologist Michael Weiss of the University of Washington in Seattle, who conducts clinical research on ALS but was not involved in this project.
Motor neurons in the brain and spinal cord decay in patients with the illness, also known as Lou Gehrig's disease. Without those neurons, muscles wither, leading to death within a few years. About 20 percent of ALS patients with the familial form of the disease have inherited a mutant form of the gene for superoxide dismutase (SOD1), which causes the protein to bind to itself, assembling into clumps suspected to contribute to the disease.
But researchers have remained puzzled as to how to link those clumps to the death of motor neurons, the hallmark of ALS. And in other neurodegenerative diseases, such as Huntington's, similar aggregations of malformed proteins actually protect against the loss of neurons. As a result, researchers have remained highly skeptical as to whether aggregates of SOD1 protein are responsible for cell death.
By watching individual cells over the course of 48 hours, Richard Morimoto at Northwestern University in Evanston, Ill., and colleagues demonstrated that most cultured neurons die between 6 and 24 hours after mutant SOD1 forms aggregates. However, cells in which mutant SOD1 remains dispersed, survive, they report.
Morimoto and his colleagues engineered cultured neuronal cells to express either the normal form of SOD1 or one of two inherited variations. They also included a gene for a fluorescent protein in order to see where SOD1 proteins accumulated within the cells. Three to five days after inserting the genes, the researchers tracked the fate of individual cells using time-lapse microscopy.
Cells with the normal form of SOD1 made the protein, but the fluorescent tag glowed uniformly throughout the cytoplasm, indicating the protein was dispersed throughout the cell. In 15 to 30 percent of cells carrying the mutant form of the protein, SOD1 accumulated around the cell nucleus. Most of the cells with aggregated proteins (88 to 90 percent) died during the observation, while most of the cells without aggregations survived, the team reports. "It is a cell-by-cell decision," Morimoto said. "If the aggregate does form, that cell is destined to die."
Morimoto suggested that the machinery for discarding misfolded proteins may have failed in some cells. Different cells may have different levels of chaperone molecules that escort unwanted molecules to the proteasome to be destroyed, he noted, and that may explain why mutated SOD1 aggregates in some cells, but not others. "Some cells may have a greater capacity to quench the misfolded protein," he said. Morimoto added that he plans to investigate how the protein aggregates may be causing cell death.
Steven Finkbeiner of the University of California in San Francisco – whose group demonstrated that aggregates can be protective in Huntington's Disease – said he is skeptical of the authors' conclusions and believes the methodology leaves room for an alternative explanation. For instance, he is concerned by the delay between when the researchers inserted the mutant gene and when they began observing the cells. Higher levels of mutant SOD1 may have accumulated in some cells and may have precipitated the aggregations the team observed, he noted. Greater or longer exposure to mutant SOD1 might be responsible for the cell death, rather than the aggregates themselves. "It is unclear to me whether [the difference between the two studies] is due to differences in the extent to which expression levels were factored in or real differences in the underlying biology," he said.
The finding may also not apply to all forms of ALS, according to Weiss, who treats ALS patients. Most ALS patients -- those with the "sporadic," rather than familial form of the disease -- have no known SOD1 mutation, he noted.