Research furthers understanding of neuron growth regulation

Experiments described in Nature Neuroscience show that neuronal development is modulated by mechanical signals linked to nerve tissue stiffness or softness, as well as chemical signals.

By Karina Toledo  |  Agência FAPESP – During the development of the nervous system, neurons grow and project axons to conduct nerve impulses along well-defined pathways. Neuroscientists have been trying for decades to discover the factors that guide this pathfinding process.

Many studies have demonstrated the importance of chemical signals in establishing neuronal pathways. Some molecules secreted by cells in nerve tissue, including the proteins ephrin and semaphorin, can push or pull axons in particular directions.

A new study, whose results have been published in the journal Nature Neuroscience, now shows that the process is also mediated by mechanical signals related to the degree of tissue stiffness.

The research was coordinated by Kristian Franze, a lecturer at the University of Cambridge in the UK, and a number of Brazilian scientists took part under the aegis of a Thematic Project supported by FAPESP.

“Our findings suggest that neurons extend axons toward softer areas of tissue and avoid stiffer areas,” Franze told Agência FAPESP. “If we succeed in understanding this mechanism better, it may be possible to find ways to modulate neuronal growth and regeneration, contributing, for example, to the treatment of spinal cord injuries and neurodegenerative diseases.”

These conclusions are based on in vitro and in vivo experiments performed at the University of Cambridge’s Department of Physiology, Development & Neuroscience. Quantitative analysis of the images recorded during the experiments was performed in collaboration with the Brazilian researchers Matheus Viana and Luciano da Fontoura Costa, both of whom are affiliated with the University of São Paulo’s São Carlos Physics Institute (IFSC-USP).

As a study model, the group opted for embryos of the frog genus Xenopus, frequently used in research on neuron growth. The first experiments were performed in vitro with embryo ganglion cells in an early stage of development.

“These cells are actually neurons located in the animal’s retina,” Franze said. “During the embryo’s development, they extend axons out of the retinas and form the optic nerves, which cross over each other to form a structure called the optic chiasm. The axons grow along this surface and loop around to the back of the head, where they connect to the brain region in which visual stimuli are processed.”

The retinal ganglion cells were cultured in a polyacrylamide hydrogel, a substrate that can be controlled for stiffness or softness. Cells cultured on soft and stiff substrates were exposed to different mechanical signals while in similar chemical environments.

“These cells grow and start to project axons in all directions. It’s impossible to analyze this with the naked eye, and at that point, we relied on the assistance of the Brazilian team,” Franze explained.

All stages of cell development were recorded by microscopy and analyzed at IFSC-USP. “We monitored neuron growth speed and curvature using ImageJ and Fiji software, among others,” Fontoura said.

The results showed that axons grew faster and were longer and straighter on the stiffer substrate, whereas on the softer substrate, their growth was slower and more exploratory, with branches crisscrossing more frequently.

The next step was to monitor neuron development in vivo. With the aid of a technique called atomic force microscopy (AFM), the group measured local stiffness in different parts of the frog embryos’ brains.

“We found different stiffness gradients in the brain tissue,” Franze said. “Neurons avoided the stiffer parts and turned toward the softer parts. We then decided to reproduce these different stiffness gradients in vitro and observed that cultured cells also grew toward the softer areas of the substrate.”

Mechanical sensor

The next set of experiments was designed to discover the mechanism by which neurons are able to perceive differences in tissue stiffness. The group chose to investigate the role of Piezo1, a protein that forms a mechanically activated cation channel in the cell membrane, resembling a pore through which ions are exchanged between the extracellular and intracellular environments.

“Piezo1 acts as a mechanical sensor for the cell. The channel opens when a certain force is applied to it. We demonstrated through in vitro experiments that the neuron growth pattern changes when this channel is blocked,” Franze said.

In vivo experiments with animals that had been genetically modified so as not to express the gene that codes for Piezo1 showed that retinal neurons did not develop adequately and were unable to reach the target region in the central nervous system.

Next, the group used AFM to put a small amount of pressure on the frog embryos’ brain tissue in order to increase stiffness at that site. Some six hours later, the neurons could be seen to be avoiding the site of increased stiffness and pursuing a different growth path.

In Franze’s opinion, these discoveries open up opportunities for the development of techniques to modulate neuron growth and regeneration, with possible therapeutic applications.

“For example, if we want neurons to grow quickly in a certain region, it should be stiff and become softer as the destination approaches to reduce growth velocity. There are several ways to change the stiffness of a tissue in vivo and thereby change the pattern of neuron growth,” he said.

More basic research is needed first, however, in order to understand more about the mechanisms of mechanical signaling. “We plan to repeat these experiments with other species of animals to see whether the findings are similar,” Franze added. “We also want to see if other kinds of nerve tissue cells are mechanosensitive like neurons. There’s still a great deal of work to do.”

The article “Mechanosensing is critical for axon growth in the developing brain” (doi: 10.1038/nn.4394) can be retrieved from nature.com/neuro/journal/vaop/ncurrent/full/nn.4394.html.