This is a simplified abstract of an article published in the Journal of Cell Biology titled: “Neuronal activity disrupts myelinated axon integrity in the absence of NKCC1b”.
In nerves outside of the brain and spinal cord — the peripheral nervous system —, myelin is made by cells called Schwann cells. We know that Schwann cells are sensitive to the activity of neurons, but we don’t understand the mechanics of this relationship.
After screening through hundreds of mutants generated in a recent genetic screen, we found a zebrafish mutant in which the myelin along the peripheral nerves is progressively disrupted. In this mutant, fluid accumulates in the space between the neuron and myelin.
An example of the mutant phenotype (bottom panel), compared to a control fish (top panel), at 7 days post-fertilisation (dpf). In these images, the myelin is fluorescently labelled in green, and the axons are fluorescently labelled in magenta. In the mutant, the myelin is severely disrupted with large fluid-filled spaces, whereas the axons appear relatively normal.
We determined that these fish had a specific mutation in the slc12a2b gene, which encodes a protein very similar to that of human NKCC1. NKCC1 is a protein that sits within the cell membrane and allows transport of electrical ions (sodium, potassium, and chloride) along with water from the outside to the inside of cells. Using CRISPR-Cas9 technology, which allows you to disrupt gene function in a very precise way, we found that the myelin pathology develops when NKCC1 function is specifically disabled in Schwann cells, and also when its function is specifically disabled in neurons. This is the first study showing that NKCC1 is an essential protein of the peripheral nervous system.
When a neuron is electrically active, it releases ions into the space between the axon membrane and the wraps of myelin — an area called the periaxonal space. It is thought that these excess ions are rapidly cleared away from the periaxonal space by channels that sit in the myelin sheath membrane, to prevent the neuron from becoming over-active. We hypothesized that NKCC1 was involved in this process. If so, its dysfunction would lead to impaired clearance of these ions following neuronal activity, and cause water to infiltrate between the axon membrane and the myelin, resulting in the observed pathology.
An electron micrograph showing a cross section of the peripheral nervous system in a control fish (left panel), and a mutant fish (right panel). In the control fish, the myelin is tightly wrapped around the axons, so the periaxonal space (shaded blue) is hardly visible, whereas in the mutant fish, the periaxonal space dwarves the axon.
To test this hypothesis, we blocked neuronal activity using tetanus toxin (the toxin that prevents neurons from communicating with each other, causing paralysis). Treating the mutants with this toxin prevented the myelin disruption, while treating mutants in which the swelling was already obvious reversed the pathology. This indicates that the peripheral myelin pathology in NKCC1 mutants occurs following neuronal activity.
In summary, our study shows that in the peripheral nervous system, NKCC1 on the myelin and/or the neuron acts to remove excess ions, to maintain normal neuronal activity.
Click on the image to play a video summary of the paper.
Read an interview with the paper’s first author, Katy Marshall-Phelps.