For release: Tuesday, May 8, 2007
In some neurological diseases, neurons might die in a surprising, yet relatively simple way: by breaking under mechanical strain.
Axons – the slender cables sent out by neurons – can be one million times as long as they are wide, and they must often traverse vast distances to reach their targets. New research on a tiny creature shows that defects in a protein called spectrin, part of a neuron's internal architecture, or cytoskeleton, could render the axon susceptible to shearing. In humans, genetic defects in a form of spectrin cause spinocerebellar ataxia type 5 (SCA5), a disease that causes uncoordinated movement and has been traced through the family tree of Abraham Lincoln.
"The idea of axon breaks is missing from the literature [on neurological disease]," says Erik Jorgensen, Ph.D., a biologist at the University of Utah in Salt Lake City. He and two colleagues at the University, Marc Hammarlund, Ph.D., and Michael Bastiani, Ph.D., have shown that a genetic deficiency of spectrin leads to axon breakage in the nematode C. elegans – a millimeter-long worm prized by neurobiologists because it has only about 300 neurons, each of them uniquely identifiable within its transparent body.
Spectrin is a filamentous protein that, in humans, exists in an alpha and beta forms, and in multiple subtypes. Until recently, most studies of the protein focused on its role in red blood cells. It forms an integral part of the cytoskeleton in those cells, creating a meshwork that helps them maintain a bi-concave shape, like a squished ball of play-dough.
Last year, researchers published a study in Nature Genetics* linking spectrin defects to SCA5 in an extended family with the disease, descended from President Lincoln's paternal grandparents. (President Lincoln, who has no living descendants, was described during his lifetime as having an irregular gait, leading to speculation that he had SCA5 himself.) The researchers, supported in part by the National Institute of Neurological Disorders and Stroke (NINDS), found that affected family members carried a mutation in the gene encoding the beta3 form of spectrin, causing reduced levels of the protein in brain cells. They also found that without enough beta3‑spectrin, proteins on the surface of brain cells appeared to become detached from their moorings, shifting to unusual locations.
Dr. Jorgensen's study on C. elegans worms, published in the Journal of Cell Biology** and supported in part by NINDS, suggests that spectrin not only helps anchor surface proteins in neurons, but also enables the cells to resist mechanical forces.
The worms he studied carry mutations in the gene for beta-G‑spectrin (the worm's nearest equivalent to human beta3-spectrin), causing them to move in abnormal patterns. The mutant worms – and normal controls – also were engineered to produce jellyfish green fluorescent protein (GFP) in their nervous system, enabling Dr. Jorgensen to view the detailed structure of neurons in live specimens. He and his colleagues specifically examined muscle-controlling neurons, which extend axons lengthwise and crosswise through the worm's body to connect with their targets.
Neurons in the mutant worms appeared to develop normally, but by adulthood, their axons were often broken. In many cases, the far end of the severed axon was still attached to a target cell. Meanwhile, the near end formed a growth cone, a fan-shaped projection that enables axons to "feel" through their surroundings, searching for their targets.
"We followed single axons through several breaks and they tried to regenerate each time," Dr. Jorgensen says.
Dr. Jorgensen and his team guessed that the mutant worms' axons break because they're vulnerable to the bending and stretching of the worm's body, and they tested that idea by paralyzing the worms. In one experiment, they bred the beta-G-spectrin mutants so that they also had a genetic deficiency of the muscle protein Twitchin, and in another, he fed the mutants a molecule that would turn off production of the muscle protein myosin. Under both conditions, the mutant worms were paralyzed, and their neurons had fewer axon breakages.
How these findings relate to the neurological symptoms in people with SCA5 is unclear. The symptoms of the disease coincide with degeneration of cells in the cerebellum – a region of the brain important for coordinating movement. Meanwhile, neurons that connect to muscles and sensory organs in the body's moving parts appear to be spared. It's possible that cells within the cerebellum are subjected to minor forces of their own making, perhaps as they extend growth cones in the developing brain, and as they attempt to make new connections in the adult brain.
"You need to label a single axon and see it break [to find out]," says Dr. Jorgensen. Such experiments will need to be performed in mice that have been engineered to contain the identical mutation found in Lincoln's family as well as complete deletions of the beta3‑spectrin gene. GFP can be used to visualize single axons in the mutant mices' brains to watch for the presence of breaks.
Meanwhile, Dr. Jorgensen plans to use his C. elegans spectrin mutants to identify other genes that affect the health of axons. The worms are ideal for these kinds of "genetic screens," since they have short reproduction times, and can be grown thousands at a time in a Petri dish. A gene that, when mutated, modifies the breakage or re-growth of axons in spectrin mutants might yield clues about how to promote axon repair in humans with SCA, traumatic brain injury, and other neurological conditions.
*Ikeda Y et al. "Spectrin Mutations Cause Spinocerebellar Ataxia Type 5." Nature Genetics, February 2006, Vol. 38(2), pp. 184-190.
**Hammarlund M, Jorgensen E, and Bastiani M. "Axons Break in Animals Lacking Beta-Spectrin." Journal of Cell Biology, January 29, 2007, Vol. 176(3), pp. 269-275.
-By Daniel Stimson, Ph.D.
Last Modified May 8, 2007