Factor Discovered to Spur Axon Growth In Upper Motor Neurons

IGF-1 Role Adds to Understanding of Nerve Cell Development and, Perhaps, Repair

The most legible signature of a corticospinal motor neuron is its lengthy axon. Perched at the top of the brain, these upper motor neurons each project a string of cytoplasm down through the spinal cord. From the cerebral cortex to the lumbar cord in humans, a single axon can be as long as a meter. The axons are a critical link in the voluntary control of our arms, legs, bladder, and bowels.

Working in mice, Jeffrey Macklis and P. Hande Ozdinler uncovered the first direct evidence that IGF-1, a growth factor that helps many neurons survive, also dramatically increases the speed and distance of axon growth in the long corticospinal motor neurons that connect the brain to the spinal cord.

By tracing the axon threads backward from the spinal cord, HMS researchers have isolated pure cultures of the neuronal cell bodies from mouse brains, regrown the long axons in the lab, and identified a potent molecule in young mice that tells the axon how fast and how far to stretch.

The findings, in the November Nature Neuroscience, represent an early step in finally understanding the biology of these developing neurons well enough to repair or replace mature neurons damaged by disease or trauma.

A Need for Speed
Corticospinal motor neurons connect the brain to the spinal cord. Their axons progressively degenerate in amyotrophic lateral sclerosis (ALS), and in related motor neuron diseases such as hereditary spastic paraplegia and primary lateral sclerosis. Damage to these neurons also underlies loss of motor function in spinal cord injuries.

Less is known about these cells than about the better studied lower motor neurons that live in the spinal cord and reach out to the muscles via the peripheral nerves (and also degenerate in ALS)—mostly because corticospinal motor neurons are embedded within the vastly more complex brain, said the study’s senior author Jeffrey Macklis, HMS associate professor of surgery and director of the Massachusetts General Hospital–HMS Center for Nervous System Repair.

Two years ago, researchers in Macklis’s lab induced apoptotic death in mouse corticospinal motor neurons and coaxed adult neural precursor cells to replace a small number of them. Immature neurons emerged, migrated to the damaged brain region, and matured into corticospinal motor neurons—eventually sending axons to the spinal cord. Some of the new neurons survived for more than a year, but most died, possibly because of the long journey (four months in adult mice) to make the necessary connections that deactivate the neuronal self-destruct program.

Since then, Macklis and his colleagues have been seeking ways to support their survival and to speed axonal elongation so the processes reach their targets sooner. According to the new paper, the researchers may have found a crucial ingredient. Insulin-like growth factor 1 (IGF-1) sets a blistering pace for axon growth of maturing corticospinal motor neurons in young mice and may have the power to do the same in older animals.

“The profound effect on neuronal cell development is an exciting observation that further validates growth factors, not only in basic biology, but as potential therapies,” said Robert Brown, HMS professor of neurology at MGH, who developed the mouse model for ALS.

Image courtesy of P. Hande Ozdinler

Swift and specific. In mice, IGF-1 accelerates the axon growth rate in corticospinal motor neurons by 15 to 20 times, akin to the tempo of early development. Other growth factors did not turn on axons, nor did IGF-1 trigger axon growth of a related type of neuron.

IGF-1 is already known for its power to promote survival of spinal motor neurons in animal models. In fact, IGF-1 injected under the skin to reach spinal motor neurons may be modestly effective in slowing down the inexorably fatal progression of ALS. Most people die from respiratory failure within three to five years of noticing symptoms. But clinical trials of IGF-1 for ALS have been plagued by equivocal results and a delivery system of questionable efficacy, Brown said. A double-blind study in the United States showed some clinical utility. At one time, the results might have validated the compound as an approved treatment for ALS, said Brown, but a second double-blind study in Europe showed insignificant outcomes, despite a positive trend. A third study under way may break the tie when it winds down next year. (Last year, the U.S. Food and Drug Administration approved two IGF-1 products for another indication—to treat very short children who are deficient in IGF-1.)

In the ALS studies, it is unclear if or how much of the growth factor reaches the peripheral nerves it is supposed to save by the subcutaneous injection used in the tests, and it would not be expected to reach the brain or corticospinal motor neurons. “The delivery is such an important variable,” Brown explained. “This paper suggests further studies are imperative. When neurons have access to IGF-1, it can be beneficial.”

Nurturing Nerve Cells
The latest study, led by postdoctoral fellow in surgery P. Hande Ozdinler, not only reports that IGF-1 is a specific and potent enhancer of axonal outgrowth but affirms the power of another growth factor, brain-derived neurotrophic factor (BDNF), to prompt treelike branching of dendrites in corticospinal motor neurons, as has been shown in several other types of neuron.

In other advances, the paper reports the first pure living cultures of corticospinal motor neurons from mice, enabling future research on potential therapeutics, interactions with other neurons and cells, and the mechanisms underlying motor neuron death. It took a year of tinkering with the timing and the culture media to consistently achieve a 10 to 20 percent overall survival rate for the purified cell cultures. “These are fragile neurons,” Ozdinler said. “But once you get them in culture as a pure population, they start talking to you. You can ask questions and get real and dependable answers from them.”

Until now, no one knew what, if any, surface markers distinguished corticospinal motor neurons from neighbors in the motor cortex. Ozdinler found the cells by injecting fluorescent microbeads into the axons and tracing them back to the midbrain. Then she purified the neurons by sorting out only the healthy glowing cells and figuring out the right culturing conditions.

Once the hard part was done, the rest of the experiments proceeded in logical sequence, like chapters in a book. Ozdinler tested candidate molecules (those with receptors on the neurons) in the cell cultures, coated beads with the molecules to observe local effects, and investigated both IGF-1 and BDNF downstream signaling cascades. She verified the importance of IGF-1 signaling in axon outgrowth of corticospinal motor neurons by blocking the receptor in mice.

In one important control, she repeated the same experiments in corticospinal motor neurons isolated from Bax-knockout mice, which carry a disrupted cell death pathway that frees the neurons from dependence on growth factors for survival. Again, IGF-1 enhanced corticospinal motor neurons’ axon outgrowth, showing that the IGF-1 role in axon outgrowth is independent of its survival function.

Movies of the dramatic effect of IGF-1 on axon growth in culture are posted on the Nature Neuroscience website with the supplementary materials.

—Carol Cruzan Morton

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