It’s spring and gardeners are out seeding, fertilizing and planting for the coming season. In the brain, another type of gardener has been working throughout the year, making sure neural networks are shaped to help their owners adapt, grow and thrive.
These gardeners, called microglia, are a type of cell that functions as an immune cell, clearing away debris and dead neurons to maintain a healthy central nervous system. They do this by a process called phagocytosis, where a cell extends processes that engulf nearby objects, swallowing them up. Scientists believe that microglia also aid healthy brain development by consuming or “pruning” parts of axons, which are the projections from a neuron that contain synapses.
Synapses are contacts between neurons that allow them to send signals from one to another. That means pruning by microglia shapes connections between neurons, determining how our brains transfer information across regions, or from our sense organs (skin, ears, nose, eyes) to the parts of the brain that process these stimuli.
Until recently, the evidence of axon pruning by microglia was indirect and never shown in live organisms (known as in vivo). For example, studies have shown that parts of axons could be found inside microglia, suggesting they had been devoured. Furthermore, when microglia are depleted, axons grow longer and make more synaptic connections. Questions also remain about how this pruning affected neural development, and whether phagocytosis is the only factor at play.
A postdoctoral researcher, Tony Lim, in the lab of Ed Ruthazer at The Neuro, decided to learn more about this phenomenon. They designed an experiment using Xenopus tadpoles, which are translucent, allowing researchers to see their brains while live. They labeled microglia with red colouring and retinal ganglion cells with green to observe their behaviour in real-time in the intact animal using a powerful microscope.
In the presence of green-dyed axons, the green-content increased in the red-dyed microglia, suggesting protein transfer from axons to microglia, a process that would happen in phagocytosis. In a few lucky cases, the investigators managed to capture footage of microglia in the act of nibbling bits off of axons!
To assess how this pruning affected development, they depleted microglial cells in tadpoles and found that this enhanced retinal axon size and altered normal behavioral responses to visual stimuli.
Cells in the body encourage microglia to nibble them by decorating their surface with a protein called complement C3, often described as an “eat-me” signal. By performing a computational search of gene databases, the scientists identified a molecule called aRCA3 that they predicted should inhibit C3. The researchers expressed (or decorated) aRCA3 on the surface of axons and confirmed that it prevented microglia from pruning the axon. From this, they concluded that complement C3 protein has an important role in targeting parts of axons for nibbling by the microglial cells.
To prove this, they expressed excess levels of complement C3 on retinal ganglion cell axons and found that this enhanced axonal pruning. This finding is especially interesting, given the fact that mutations in the human complement pathway are among the strongest genetic predictors of schizophrenia susceptibility, suggesting a possible link between phagocytosis of axons by microglia and neurodevelopmental disorders like schizophrenia and autism.
The study shows the first direct, in vivo evidence demonstrating that microglia prune axons, which promotes proper neural wiring during development. In this model, neurons signal the need for pruning by expressing regulatory proteins. Microglia then go and do the gardening.
“For years, developmental neuroscientists have been suggesting that the immune system has an important role to play in healthy brain development,” says Ruthazer. “Tony’s study directly implicates the brain’s own immune cells, microglia, in the act of shaping axonal projections, and reveals one possible mechanism by which genetic mutations in the complement pathway may lead to certain neurodevelopmental disorders.”
"Microglial trogocytosis and the complement system regulate axonal pruning in vivo", by Tony KY Lim and Edward S Ruthazer, was published in the journal eLife on March 16, 2021. Click here for the full study.