Roughly 60 million people worldwide have epilepsy, a neurological condition characterized by seizures resulting from excessive neural activity. Using a fruit fly model of epilepsy, where seizures result from neurons that are vulnerable to becoming hyperactivated by stress, new research has identified a key sequence of molecular events in this process.

The research, from the lab of Troy Littleton, PhD, professor of biology at the Picower Institute for Learning and Memory at the Massachusetts Institute of Technology, is published in eLife in a paper titled “Glial Ca²⁺ signaling links endocytosis to K+ buffering around neuronal somas to regulate excitability.”

The research team had previously characterized a Drosophila temperature-sensitive mutant termed zydeco (zyd) that exhibits seizure-like behavior when exposed to a variety of environmental stressors. The zyd mutation disrupts an NCKX exchanger that extrudes cytosolic Ca²⁺. The lab used this fly model to better understand how altered cortex glial Ca²⁺ signaling regulates neuronal excitability.

The team conducted an unbiased, in vivo RNAi screen for modifiers of the seizure-like phenotype in Drosophila. The result was evidence for a direct link between glial Ca ²⁺ signaling and classical functions of glia. More specifically, that chronic elevation of glial Ca ²⁺ causes hyperactivation of calcineurin-dependent endocytosis, leading to an endo-exocytosis imbalance.

The zyd mutation creates a protein that helps to pump calcium ions out of the cells and is specifically expressed by cortex glial cells. But that didn’t explain why a glial cell’s difficulty maintaining a natural ebb and flow of calcium ions would lead adjacent neurons to become too active under seizure-inducing stresses such as fever-grade temperatures or the fly being jostled around.

The activity of neurons rises and falls based on the flow of ions—for a neuron to “fire,” for instance, it takes in sodium ions, and then to calm back down it releases potassium ions. But the ability of neurons to do that depends on there being a conducive balance of ions outside the cell. For instance, too much potassium outside makes it harder to get rid of potassium and calm down.

The need for an ion balance—and the way it is upset by the zydeco mutation—turned out to be the key to the new study. The team found that excess calcium in cortex glia cells causes them to hyper-activate a molecular pathway resulting in the withdrawal of many of the potassium channels that they typically deploy to remove potassium from around neurons. With too much potassium around, neurons can’t calm down when they are excited, and seizures ensue.

“No one has really shown how calcium signaling in glia could directly communicate with this more classical role of glial cells in potassium buffering,” Littleton said. “So this is a really important discovery linking an observation that’s been found in glia for a long time—these calcium oscillations that no one really understood—to a real biological function in glial cells where it’s contributing to their ability to regulate ionic balance around neurons.”

In order to explore potential treatments, Shirley Weiss, PhD, a postdoc in the Littleton lab and first author on the paper, interfered with expression in 847 potentially related genes and found that about 50 affected seizures. Among those, one stood out both for being closely linked to calcium regulation and also for being expressed in the key cortex glia cells of interest: calcineurin. Inhibiting calcineurin activity, for instance with the immunosuppressant medications cyclosprorine A or FK506, blocked seizures in zyd flies.

Weiss then looked at the genes affected by the calcineurin pathway and found several, one of which, sandman, led to seizures in the flies when knocked down. Further research showed that hyperactivation of calcineurin in zyd glia led to an increase in endocytosis in which the cell was bringing too much sandman back into the cell body. Without sandman staying on the cell membrane, the glia couldn’t effectively remove potassium from the outside.

When Weiss and her co-authors suppressed endocytosis in zydeco flies, they were able to reduce seizures because that allowed more sandman to persist where it could reduce potassium. Sandman, notably, is equivalent to a protein in mammals called TRESK.

“Pharmacologically targeting glial pathways might be a promising avenue for future drug development in the field,” the authors wrote in eLife.

In addition to that clinical lead, the study also offers some new insights for more fundamental neuroscience, Weiss said. While zyd flies are good models of epilepsy, Drosophila’s cortex glia do have a property not found in mammals: they contact only the cell body of neurons, not the synaptic connections on their axon and dendrite branches. That makes them an unusually useful testbed to learn how glia interact with neurons via their cell body versus their synapses. The new study, for instance, shows a key mechanism for maintaining ionic balance for the neurons.