“Nature has come up with this beautiful symphony of molecular signaling that allows a tissue as incredibly complex as the brain to wire appropriately. We’re learning more and more about how this happens, but we don’t yet have the ability to direct all these intricate processes,” said James Harris, a graduate student in the Arlotta lab and lead author of the study. “Instead, we created an extremely precise tool that allows us to override the molecular signals within the body and guide axonal growth, according to our own designs.”

By controlling axonal growth directly, this strategy avoids disrupting critical biological signaling molecules or introducing chemicals that might alter the delicate developmental environment, which could potentially cause unintended consequences on neighboring cells. To make sure the tool was highly specific, the researchers took an engineering approach to the problem.

A noninvasive engineering approach

To control axon growth, the researchers introduced a fusion protein into specific neurons that combined the functionality of two different proteins. The first protein is normally expressed in developing axons, and controls the machinery responsible for axonal outgrowth. The second protein is originally found in plants and helps them to sense light.

“Much in the way that plants grow toward the sun, we engineered the axons so they grow toward our targeted illumination,” Harris said.

When the researchers shone a specific type of light near the neurons, the axons grew toward the noninvasive stimulus.

The researchers tested the approach in a zebrafish model, in collaboration with the Leonard Zon lab. They were able to not only make the neurons grow in a specific chosen direction, but also make the neurons grow across repulsive developmental barriers that normally restrict axons to a very narrow body location.

“There are specific molecules that are expressed in these developmental barriers that help guide axons correctly during normal development. Interestingly, many of these molecules are also present in damaged tissue and act as barriers to axonal regeneration in mammals,” Harris said. “In this particular context of a developing zebrafish embryo, our approach had the power to overcome these inhibitory signaling molecules.”

The researchers also studied a zebrafish model with genetic mutations that prevented axons from growing correctly. Their illumination approach successfully rescued this defect by guiding axons to their targets. The guided axons were able to cause muscle contractions in the zebrafish, demonstrating that the repaired connections were functional.

Applying the technology

Although applying this technology to repairing injured connections in patients will require substantial additional work, this study is a promising step in this important direction. More immediately, the new technique can help scientists create more accurate models of the brain.

“We are really interested in using this technology to wire more specific connections within human brain organoids,” Arlotta said.

Made from human stem cells, organoids replicate important features of the developing nervous system in a lab dish.

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“Brain organoids can reproducibly make a large number of cell types that normally populate the endogenous brain. Although these neurons can extend axons and wire within circuits, at the moment, their connectivity is not organized like that of the actual brain,” Arlotta said. “By guiding axons of specific neurons to pre-defined targets, we would have an opportunity to engineer new connectivity, exactly as present in the intact organism. This is important for many reasons, not the least the possibility of understanding how diseases affect specific neurons and their networks to inform therapeutic progress.”

This study was supported by the National Institute of Neurological Disorders and Stroke, the National Institute of General Medical Sciences, the National Institutes of Health, the Harvard Center for Biological Imaging, and the Harvard Stem Cell Institute.