Molecule may be key to spinal cord regeneration

Researchers at the Queensland Brain Institute and the University of Melbourne have identified a molecule which they believe may play a crucial role in preventing spinal cord regeneration.

The molecule EphA4, a receptor tyrosine kinase, is normally expressed during the development of the nervous system, and appears to play a role in correctly guiding spinal cord axons to their eventual targets by blocking growth in inappropriate directions.

But the researchers, led by Prof Perry Bartlett at QBI and his former post-doc Anne Turnley, now at the University of Melbourne's Centre for Neuroscience, and the School of Physiotherapy's Prof Mary Galea, have shown in EphA4 knockout mice, that the molecule may be a key player in the inhibition of spinal cord regeneration.

The results of their studies have been published in the Journal of Neuroscience.

When the spinal cord is damaged, the injured axons retract from the site of injury, before starting to regrow. But the regenerating axons usually don't make it past the site of injury, let alone to their original targets, suggesting that there is an inhibitor or inhibitors blocking regeneration.

A second impediment to regrowth is the process of astrocytic gliosis, where the astrocytes present around the site of injury activate, becoming hypertrophic, and causing glial scarring, which physically blocks regrowth of the axons. The process also includes secretion of a variety of cytokines and other molecules including cell adhesion molecules and extracellular matrix molecules.

Bartlett and Turnley's project began with the creation of an EphA4 knockout mouse used to study the development of the spinal cord by student Yona Goldschmit, now a postdoc with Turnley.

"A lot of these receptor tyrosine kinases are involved in growth and mapping of axons, so we knew it was an interesting molecule," Bartlett said. As it happened, the knockout mice had a peculiar hopping gait.

"It turned out that many of the axons down the spinal column when to the wrong place, and didn't find the appropriate hook-ups. We reasoned that if the molecule was so important in promoting growth in the spine, then it could be important in preventing repair of spinal cord damage."

So the researchers embarked on a series of complex experiments to examine spinal cord injury in the knockout mice. To their surprise, the animals not only regained the ability to walk, but grasp and climb at the levels normal to the knockout phenotype.

"It was real regeneration, real regrowth, and not spontaneous walking caused by spinal reflexes," Bartlett said. "We tested it rigorously over several months."

Even more remarkable was the fact that the typical astrocytic gliosis response to spinal cord injury was also significantly down regulated in the knockout mouse. The spinal cords of the injured knockouts regenerated so well, there was no sign of the lesion at all, with 70 per cent of the axons finding their way back through the injured site to their target.

In vitro experiments demonstrated that EphA4-expressing astrocytes were able to block axon growth. Further experiments showed that EphA4 expression by astrocytes was regulated by inflammatory cytokines such as gamma interferon, and in turn regulated the downstream responses that causes glial scarring. With no EphA4 expression in the EphA4 knockout mice, the astrocytes were not activated by the inflammatory response to injury and very little scarring resulted.

"We think EphA4 is the predominant molecule that regulates the astrocyte activation response," Bartlett said. "It may be the key regulator."

The researchers are also pretty confident that a similar mechanism is at play in human spinal cords, with preliminary evidence from primates suggesting that EphA4 plays the same role.

The discovery has major implications for the treatment of spinal cord regeneration, suggesting a variety of therapeutic approaches targeting EphA4 expression, as well as the specific inflammatory responses to injury that lead to its activation. While broad anti-inflammatories such as corticosteroids have not been shown to be useful in the treatment of spinal cord injury, a more targeted approach may prove to be a better option.

"The real test will be to show in a wild-type animal that blocking EphA4 allows spinal cord regrowth," Bartlett said. "We hope to have data on blocking by next year, and to have identified the best approach to blocking activation in the next couple of years."