Periodic Reporting for period 1 - LAMININ (Light-switchable proteins and Adhesion Micropatterns to Illuminate the Navigation machInery of Neurons) Reporting period: 2020-04-01 to 2022-03-31 Summary of the context and overall objectives of the project The central nervous system (CNS), which is composed of the brain and the spinal cord, controls most of the functions of the human body and mind, including voluntary and involuntary movement, and is responsible for our thoughts, perceptions and emotions. The basic functional units of the nervous system are neurons (nerve cells). These electrically excitable cells communicate with other neurons through specialized connections called synapses. The CNS is composed of an extensive network of ~86 billion connected neurons organized into specific interconnected regions with defined functions. Neurons form connections via long extensions that send out (axons) or receive (dendrites) electrical signals. The establishment of connections over large distances between different brain regions, relies on precise pathfinding capabilities of individual neurons during embryonic development. For example, callosal axons that connect the two halves of the brain (via a thick nerve tract, called the corpus callosum), must navigate a complex trajectory to reach all the way across the brain. Navigation relies on a structure at the tips of neuronal extensions, termed the growth cone, which is able to detect a variety of guidance molecules (such as netrin) that function like traffic cops to guide axons and dendrites to their targets. Dysfunction of neuronal navigation has been linked to numerous neurodevelopmental disorders such as Hirschsprung’s disease, Kallmann syndrome, ACC as well as autism spectrum disorders and epilepsy, and is frequently associated with mutations in genes that encode known navigation machinery components. However, in the majority of cases, the causes of these disorders are unknown, and this is in part because we lack a complete mechanistic understanding of this complex process. Improving our understanding of neuronal guidance will not only enable more accurate diagnosis of these disorders, it could also improve our ability to develop more accurate organ-on-chip models. Objectives of this Marie Skłodowska Curie Action (MSCA) have been to (a) identify the protein components of the neuronal navigation machinery that sense and respond to netrin signals, (b) to determine where these proteins localize within actively moving neurons, and (c) to develop new ways to control the navigation machinery, and neuronal navigation. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far In order to construct a map of the main components of the navigation machinery that controls attraction and repulsion to netrin signals we isolated netrin receptor complexes from cells, with the goal to isolate receptors plus any interaction partners (other navigation machinery proteins). Because these binding partners are to a large extend unknown, we used a protein identification technique called mass spectrometry to identify these proteins. We identified multiple proteins that were not previously recognized as components of the navigation machinery. To study these proteins in more detail we performed microscopy experiments in which we labelled these proteins with a fluorescent tag in living neuronal cells to study their localization and dynamics. We found that several of the identified proteins localized precisely at the same position as the netrin receptors, suggesting that these are indeed components of the navigation machinery. To understand how the location of netrin receptors and binding proteins changes during navigation, we developed a microscopic assay to track the movement and navigation of hiPSC derived neurons, over long distances and timescales (up to several days) by using custom designed cell adhesion micropatterns (to which neurons can stick) that we generated in the lab. This allowed us to observe neuronal movement and navigation in great detail, including the processes that occur inside neurons, such as changes in the localization of the netrin receptors and interactors, and the assembly and disassembly of the cellular skeleton (which drives cell movement). Finally, this project aimed at the development of new tools to precisely control netrin signaling, and thereby neuronal navigation. We succeeded in constructing a light controllable variant of one of the netrin receptors. This strategy works exceptionally well, and our results indicate that activation works as well as netrin (ligand) mediated activation, but with the added advantage that we can much more precisely control it, by using light patterns. We are currently performing experiments to determine how localized receptor activation causes changes in navigation direction. Furthermore, we are currently testing if we can instruct neurons to move in a precisely defined manner using micropatterns, and ultimately we aim to employ this technology to build complex networks of neurons, e.g. for use in the development of more advanced organ-on-chip models.Overview and dissemination of results• We identified several new components of the neuronal navigation machinery, through an innovative protein interaction screen, combined with high resolution fluorescence microscopy of the identified components in actively moving neurons• Results have been communicated to collaborators and local stakeholders in regular internal meetings and workshops. Results will be communicated to the scientific community through publication in scientific journals.• Novel micropatterning tools can be used to standardize neuronal navigation assays, and enable high resolution imaging of neuronal navigation events.• Results have provided new teaching materials for M.Sc students in Molecular Medicine and Neuroscience.• Light controllable variants of neuronal guidance receptors were developed, that allow for precise spatial control of guidance signaling, which will enable more precise dissection of the neuronal guidance machinery • Results have been communicated to a scientific audience through poster and oral presentations at (inter-)national conferences and meetings. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) To summarize, we have identified several new components of the netrin pathway for neuronal navigation, through an innovative protein interaction screen, which we envision can be utilized for many other guidance receptors. We verified that various of these interactors co-localized with netrin receptors, and tracked their dynamics in living neurons. We are now exploring what the consequences are of the inactivation of these interactors for navigation. Furthermore, we developed an in vitro navigation assay using a protein micropatterning setup that allows us to direct neurons along specified trajectories, thereby enhancing our ability to visualize directional steering events in great detail, and allowing us to directly study the protein machinery that mediates guidance. This assay could form the basis for studying directional migration of various types of neurons, that are attracted by different types of guidance signals, in a highly standardized way. Finally, we developed light activated variants of netrin receptors that can be activated almost instantly in living cells, and we are currently testing how localized activation of guidance pathways affects directional steering. In the future, through further development of this combinatorial approach we aim to improve our understanding of neuronal guidance, which will enable more accurate diagnosis of neuronal guidance related disorders, and finally, new technologies that were developed in this proposal could aid in the development of novel organ-on-chip technology. How do neurons find their way in the developing brain?