A molecule responsible for the proper formation and function of neurons finds its way to the right place not because it is actively recruited, but because it can't go anywhere else.

Researchers at Baylor College of Medicine have identified a distal axonal cytoskeleton that functions as a boundary to ensure a key scaffolding protein, called ankyrinG, stays at the start of the axon near the cell body where it performs its functions of clustering sodium and potassium ion channels and maintaining the neuron's proper organization (polarity). The findings appear in the current edition of the journal Cell.

AnkyrinG essential to axon development

"It has been known that ankyrinG is required for the axon initial segment to form. The axon initial segment is the 'gatekeeper' of all neuronal output, and is responsible for the decision of whether a signal is sent on to the next neuron in the circuit or not. Without the axon initial segment, there would be no output of information," said Dr. Matthew Rasband, associate professor of neuroscience at BCM, and senior author on the project. "Every known protein found at the axon initial segment depends on ankyrinG. If ankyrinG is not clustered at the proximal part of the axon, then the axon initial segment doesn't form and the neuron doesn't fire."

Position determined by barrier inside axon

Rasband said the big question was, "How does ankyrinG reach its proper place in the neuron?" Members of the Rasband laboratory began by analyzing when and where the axon initial segment forms in the developing brain. They found that ankyrinG always appeared in exactly the same location during development.

"AnkyrinG would start to enter into the axon and then it was almost as if it hit a wall and couldn't go any further," Rasband said. "This observation suggested to us that there was some type of boundary or barrier restricting ankyrinG to the start of the axon where it could then assemble the axon initial segment."

Moving the boundary

To further study the properties of the boundary, they developed methods to disrupt or move the boundary, and to test the effects of these manipulations on the clustering of ankyrinG in the axon. In both cultured neurons and mouse models, they were able to move the boundary to different locations along the axon. Doing this allowed researchers to change the length of the axon initial segment.

If the boundary was farther away from the cell body, then the length of the axon initial segment was longer. If the boundary was closer to the cell body, then the length of the axon initial segment was shorter. When they removed the boundary all together, ankyrinG would not cluster at the start of the axon and the axon initial segment failed to form.

Implications for disease

"We had anticipated that some molecule recruited ankyrinG to the axon initial segment, but instead, and much to our surprise, we found a barrier that excludes it from the distal axon, effectively restricting it to the location where the axon initial segment should be," Rasband said. "These results have important implications because they imply a similar exclusion mechanism might be in play or functioning not only at the axon initial segment, but all of the places where ankyrinG is found, including cardiac cells, at nodes of Ranvier in myelinated axons, muscle cells, and epithelial cells."

Rasband said that the axon initial segment is emerging as a key player in nervous system disease and injury, with newly discovered mutations in genes encoding axon initial segment proteins (including ankyrinG) implicated in diverse diseases including autism, epilepsy, schizophrenia, and bipolar disorder. Understanding how ankyrinG functions to assemble and maintain the axon initial segment could one day play a role in finding treatments for these diseases.

Contributors to the study include: co-first authors Drs. Mauricio Galiano and Smita Jha, and Chuansheng Zhang, all of whom are postdoctoral associates in neuroscience in the Rasband laboratory; Dr. Yasuhiro Ogawa, a former postdoctoral associate in the Rasband laboratory and currently an assistant professor at Meiji Pharmaceutical University Tokyo, Japan; Tammy Szu-Yu Ho and Kae-Jiun Chang, both graduate students in the Rasband laboratory and the program in developmental biology at BCM; Dr. Michael C. Stankewich, Yale University; and Dr. Peter J. Mohler, Ohio State University Medical Center.

This study was supported with funding from the National Institutes of Health, the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation, Mission Connect, and the Eunice Kennedy Shriver National Institute of Child Health and Human Development.