Now new studies by a Canadian research team and Japanese collaborators, with support from the Packard Center for ALS Research at Johns Hopkins, strengthen earlier theories that whatever ultimately tips motor neurons onto their downhill path likely comes from outside the cells. The work describes a mechanism in which a warped, toxic molecule is secreted from cells into a motor neuron's environment, where it harms the neuron.

The studies, described in this month's issue of Nature Neuroscience, could focus therapy research on easier-to-access areas outside of cells.

Led by Packard scientist Jean-Pierre Julien of Quebec's Laval University, the work is largely based on mouse models of ALS - animals engineered with a mutant human gene responsible for a familial form the disease. Mice carrying the mutant SOD1 gene develop ALS and die. Those with normal SOD1 are fine.

In some earlier, rather exotic studies where only some mouse cells were made to carry mutant SOD1 genes, Julien's team showed, to their great surprise, that even though motor neurons may carry mutant genes, they apparently don't die as long as neighboring cells are normal. And, conversely, having neighbors with mutant SOD1 can trip motor neuron death even if the motor neurons themselves have the undamaged version. Other Packard scientists have confirmed the unusual results that say a motor neuron's surroundings are all-important in developing disease-what they call the "bad neighborhood" theory.

In the new studies, Julien's team found that misshapen, mutant SOD1 is preferentially "booted out" of nervous system cells - secreted by motor neurons and other neighboring cells - into cell surroundings. Once there, the researchers show, the mutant SOD1 is directly toxic to motor neurons it encounters. Mutant SOD1 can also rouse nearby immune cells, the microglia, to release neuron-harming agents.

The team's studies began with a fishing expedition of sorts, using a yeast two-hybrid approach, a modern technique that uses a cell protein as "bait" to see what other molecules associate with it. Mutant SOD1 was consistently linked with chromogranins - molecules closely tied to cell-secretion systems. In both nerve-cell cultures and in the spinal cords of the model mice, the team observed mutant SOD1 - but not normal SOD1 - paired with chromogranins, Further, the scientists found the pairings throughout a cell's secretory machinery, a "packaging" system called the trans-Golgi network. The older the mice, the more paired molecules appeared. "We believe that chromogranins chaperone the mutant SOD1, helping it through a cell's secretory pathways," says Julien.

Last, the researchers discovered that chromogranin production, and, accordingly, mutant SOD1 secretion, is dramatically stepped up in cells adjacent to motor neurons, bathing them in the misshapen molecule. "In a motor neuron that's under siege from events within," Julien says, "having toxic molecules outside may be a last straw in the cell's coping ability. All this," he says, "becomes a part of ALS pathology that hasn't been explored."

The research was supported by the Robert Packard Center for ALS Research at Johns Hopkins, the ALS Association, the Canadian Institutes of Health Research, the ALS Society of Canada, the Japan Society for the Promotion of Science and Japan Foundation for Neuroscience and Mental Health.

Scientists participating in the study are Makoto Urushitani (the first author) of Laval University and Attila Sik, Takashi Sakurai, Nobuyuki Nukina and Ryosuke Takahashi of the RIKEN Brain Science Institute in Saitama, Japan.

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