There are 100 billion neurons in the brain, and each of them forms thousands of connections with other neurons. Despite this immense complexity, neural connections are established in a highly specific manner to build functional circuits. The molecular mechanisms underlying synapse formation are therefore intricate and finely tuned. Key players in this process are cell adhesion molecules (CAMs), which assemble into trans-synaptic complexes across the synaptic cleft to link two neurons. Understanding the molecular features that determine their compatibility is crucial for elucidating neuronal recognition before synapse formation.

In the first chapter, I provide a more detailed introduction to the importance of specificity in neural connectivity and discuss the role of CAMs in this process. I then explore two protein-modifying mechanisms that expand the diversity beyond the genetic code: glycosylation and alternative splicing. Examples of how these mechanisms regulate CAM function at the synapse are presented. Finally, I introduce Teneurins, a family of synaptic CAMs, and discuss their roles in organizing brain circuits across different animal models.

In the second chapter, I describe a pulldown protocol specifically optimized for CAMs to establish their interactome from mouse brain tissue. This protocol significantly reduces the amount of animal material required. Representative data are presented using Teneurin-3 as bait, providing an overview of its interactome. I also examine the effects of testing different detergents during brain lysate preparation and of applying different normalization strategies to the resulting mass spectrometry data - two methodological aspects rarely discussed in similar studies. An extensive, user-friendly protocol for brain lysate preparation, pulldown, and data analysis is provided in the supplementary methods.

In the third chapter, I investigate how alternative splicing of Teneurin-3 influences its interactions with other CAMs. I first present results from mouse brain pulldowns (as detailed in Chapter 2) using the four splice variants of Teneurin-3 as bait. Next, I apply the AlphaFold Multimer pipeline to predict the likelihood of interactions between these variants and potential binding partners. Finally, I experimentally test the predicted interactions using a cell-clustering assay, which specifically measures the ability of two proteins to form trans-synaptic complexes. These results reveal an alternative-splicing dependent mechanism regulating Teneurin-3 interactions.

In the fourth chapter, I systematically characterize the effects of Teneurin-3 N-glycosylation on its expression and stability. To this end, I generated a glycomutant library of Teneurin-3 extracellular domain constructs, each carrying a mutation at a distinct N-glycosylation site that prevents glycan attachment. These data are compared to mass spectrometry-based characterization of the Teneurin-3 glycosylation profile, which identifies the types of glycan chains present at individual sites. Beyond providing structural insights, the establishment of this library offers a strong foundation for future research.

In the fifth chapter, I address the unusual N-glycosylation profiles typically observed for brain proteins as an emerging issue in understanding the molecular basis of synapse formation. Drawing on recently published data, I propose that this atypical glycosylation profile may shift following synaptic activation, acting as a molecular mechanism that helps stabilize functional synapses, including through effects on CAMs.

Membrane proteins are of vital importance for human life. They reside in the plasma membrane, where they provide the adhesive forces needed for cells to spatially organise themselves relative to one another. Additionally, given their unique localisation - being in contact with both sides of the membrane - membrane proteins enable various modes of sensing and transmembrane signalling. For instance, channel proteins regulate the selective membrane permeation to ions and nutrients. Various other membrane proteins transduce information across the membrane by structural changes in response to direct binding interactions on either the intra- or extracellular side.

In the first chapter, we briefly introduce the role of a particular type of membrane proteins, namely synaptic cell adhesion molecules (synCAM) and their importance in organising neuronal networks. We discuss the role of synCAMS in recognizing extracellular cues which constitutes the mechanistic basis for network formation, and focus on the structural basis of synCAM binding specificity.

In the second chapter, teneurin-3 (Ten3) is investigated as a model synCAM for splicing-dependent binding specificity. We resolve the structures of various isoforms to reveal how small splice inserts completely reorganise intramolecular domain configuration. These structural changes are shown to underlie trans-cellular clustering and the guidance of axon outgrowth.

In the third chapter, we investigate the molecular basis of a neurological disease called microphthalmia ("small eye disease"). A unique patient-derived Ten3 missense mutation resides at a intramolecular domain-domain interface specific to two of the splicing isoforms. We showhowthe point mutation disrupts trans-cellular binding which may explain the eye and brain malformations, and thereby the cognitive impairments typical inmicrophthalmia patients.

In the fourth chapter, we look into the structural basis of glycosylation-dependent binding specificity between synCAMs contactin 1 and neurofascin 155. Only specific glycan chains at the interface allow formation of the complex, and subsequently, enable the trans-cellular contactin 1 - neurofascin 155 interactions.

In the fifth chapter, we set up a workflow for the reconstitution of purified neuronal membrane proteins in the lipid bilayers of vesicles over which transmembrane voltages can be induced. This will allow structural studies of neuronal membrane proteins in an environment more representative of the neuronal membrane, which is subjected to the dynamical voltages of the action potential.

In the sixth chapter, we provide an overview of techniques that have enabled the reconstruction of small, low signal-to-noise proteins using electron cryo-microscopy (cryo-EM). These approaches typically involve appending additional protein domains to the target, thereby facilitating particle picking and alignment needed for high-resolution cryo-EM single-particle analysis. This methodological review is aimed to select an approach for reconstructing the membrane-embedded proteins of chapter 5.

The central nervous system has a very limited capacity to regenerate damaged tissue. Therefore, regeneration strategies focus on transplantation of neural stem cells or differentiated neural cells. In order to make such a treatment effective, it is important to understand the mechanisms that enable cell differentiation. It is well known that besides biochemical cues, also mechanical and geometric properties of the cell environment, such as topography, curvature and stiffness, can influence the process, which has been studied mostly in 2D. In order to conduct relevant cell studies in vitro, it is therefore important to mimic the 3D structure of the in-vivo cell environment. Many different approaches have been adopted to create scaffolds for neuronal cells, such as freeze-drying, electrospinning and stereolitography. The main drawbacks of these methods are the limited resolution and the constraints in terms of achievable geometries. Two-photon polymerization overcomes these problems by using a laser to polymerize a photosensitive material in extremely confined volumes, achieving a submicrometric resolution. In this study, we fabricated 3D microscaffolds made of an acrylate polymer called IP-Dip by employing two-photon polymerization in order to study the effect of curved versus straight lattice geometries on the differentiation of mouse embryonic stem cells into neural progenitor cells. First, feasibility studies were carried out with HeLa cancer cells and the effect of curvature on these cells was investigated on 2.5D structures. We established a workflow for conducting these experiments from the fabrication up until the analysis. By employing confocal imaging, image stacks were obtained and then analysed to obtain the volumetric cell occupancy of the scaffolds and identify the location of the cells within the scaffolds. We concluded that mESCs could successfully grow and differentiate within the 3D scaffolds without a specific preference for a curved over a straight lattice structure.