A team of Israeli researchers has revealed a mechanism that participates in the navigation of neurons in the brain

The research shows how the Robo signaling system works and affects the developmental processes in the brain, as well as outside the central nervous system, and will help in the development of new drugs to treat cancer, chronic kidney disease and macular degeneration

During the development of the brain, billions of nerve cells (neurons) must find their exact place in order to promote the creation of trillions of correct connections between neurons, thus allowing us to enjoy normal motor, perceptual and emotional functions.

To achieve such a high level of precision, neurons express special protein receptors that sense the environment and serve as 'navigators' directing the neurons and the long processes that come out of them along the correct paths while avoiding entering forbidden areas.

In a new study published in the scientific magazine Cell, researchers from Bar-Ilan University, along with several colleagues and collaborators, report the discovery of a sensitive molecular mechanism that gives the neural receptor 'Robo' the ability to respond to signals in its environment in a specific way.

One of the most important signaling systems in neuron navigation processes includes the 'Robo' receptor that is displayed on the surface of the navigating cells and the extracellular protein 'Slit' that binds to 'Robo' and activates it. Genetic defects in one of these two proteins lead to serious damage to the structure and function of the brain. For example, a deficiency in 'Robo' or 'Slit' impairs the brain's ability to form appropriate connections beyond the area of ​​the brain called the 'corpus callosum'. Through this area, nerve cell extensions from one half of the brain (hemisphere) cross the path of the extensions that come out of the other hemisphere on their way to innervate the organs on the opposite side of the body, a very important feature in animal physiology.

"Uncontrolled activity of Robo and Slit contributes to the formation and development of a number of chronic and developmental diseases such as macular degeneration, bone depletion, and chronic kidney diseases," explains Prof. Jordan Optovsky, the editor of the study and the head of the Structural Biology Laboratory at the Faculty of Life Sciences "S. Mina and Orred Goodman at Bar-Ilan University.

Also, there is a special interest in the involvement of Robo and Slit in cancer. Normally, the cells in our bodies receive signals from their environment that direct them to growth, division, differentiation, migration and ultimately programmed cell death. These signals are received through receptors displayed on the surface of the cells that transmit the desired information into the cell. In cancer, some of these receptors are "enslaved" to allow the appearance and creation of tumors and the sending and establishment of metastases.

In personalized medicine, based on the analysis of the genetic profile of the cancer, drugs are used that block the activity of those recalcitrant receptors, thus preventing the continued proliferation of the cancer cells. "Robo and Slit appear uncontrollably in cancer and for some time researchers have considered these proteins as attractive targets in the treatment of incurable cases of pancreatic, skin and breast cancer," explains Prof. Optovsky. "The problem is that currently there are no drugs that aim to damage the activity of Robo and Slit, and we hypothesized that the reason for this is a lack of structural understanding regarding the activation and signaling mechanism of Robo. Our discoveries provide, for the first time, the knowledge needed to design effective drugs aimed at controlling Robo's activity in patients, thus expanding the range of possible treatments in the fight against cancer.'

You can see in Slit and Robo a very high level of evolutionary conservation, and you can find this pair of proteins in almost all living things that include a nervous system, from capillary worms (nematodes) a single millimeter long to humans, which indicates their great importance for the functioning of the nervous system. Using X-ray crystallography, Prof. Optovsky, doctoral student Reut Barak and their partners were able to decipher the spatial structure of the Robo receptor at the level of atomic separation. "The crystallographic work and deciphering the structure took about six years, but this was only the first stage of the research work," says Prof. Optovsky. "The atomic structures showed us quite clearly how two Robo receptors connect together to form an active dimer, and how the contact points that participate in dimerization are blocked in the inactive receptors." The structural discoveries formed the basis for a series of further biochemical and developmental experiments carried out by the laboratory director Dr. Valia Gaz-Hadad and the doctoral student Galit Yom-Tov.

Since discoveries about two decades ago, great progress has been made in understanding the biological roles of Slit and Robo and the developmental processes they are responsible for in the brain, as well as outside the central nervous system. However, it is still unknown how Slit activates Robo and how the latter maintains itself in an inactive state in the absence of Slit.

Relying on the new structural knowledge, Prof. Optovsky's research group developed a new biochemical assay with which they followed the dimerization of Robo in cell culture. In this way, they supported the structural model according to which Robo controls its activation through a self-inhibiting mechanism that prevents dimerization in the absence of Slit, and showed that the same mechanism is also present in several Robo-like receptors. In the next step, the research group focused on examining the new mechanism in the system of a living organism, as explained by Prof. Optovsky: "We took a narrow research approach and followed the elongation of one type of neuron. During evolution, the genes coding for Slit and Robo were duplicated several times, so that in mice and humans there are four Robo and three Slit proteins, each with unique aspects but also some functional overlap. It was clear to us that if we perform the planned experiments, where we make targeted genetic changes, in a model animal with a large number of Robo receptors, it will be difficult to discern differences in biological activity due to a compensatory effect from the other receptors. Fortunately for us, in the tiny capillary worm C. elegans, only a single gene for Robo and one for Slit has been preserved, which somewhat simplifies the experiments as well as the analysis of the results." Thus, with the help of Prof. Sivan Kornblit-Hanis, also from the Faculty of Life Sciences at Bar Ilan University, and an expert in the genetics and development of C. elegans, a series of axon targeting experiments were conducted that confirmed the structural model and the biochemical results.