The three proteins Teneurin, Latrophilin and FLRT hold together and bring neighboring neurons into close contact, enabling the formation of synapses and the exchange of information between the cells.
In the early phase of brain development, however, the interaction of the same proteins leads to the repulsion of migrating nerve cells, as researchers from the Max Planck Institute of Neurobiology and the University of Oxford have now shown.
The detailed insight into the molecular guidance mechanisms of brain cells was possible due to the structural analyses of the protein complex.
Well anchored, the proteins Teneurin and FLRT are located on the surface of nerve cells. They are on the lookout for their partner protein, Latrophilin, on other neurons. When the three proteins come into contact, they interconnect and hold the membranes together.
They then trigger still largely unknown signaling cascades and thus promote the formation of a synapse at this site.
Teneurin and its partner proteins are known to establish these important cell contacts in the brain. Teneurin is also an evolutionary very old protein, with related proteins found in diverse organisms ranging from bacteria to worms, fruit flies and vertebrates. However, the role of these proteins during brain development, when neurons are not yet forming synapses, remained unknown.
Studying the function of the protein complex
An international team of researchers now investigated in detail the structure of the Teneurin-Latrophilin protein complex. Using high-resolution X-ray crystallography, they were finally able to find out more about its function in early brain development.
The structural analyses and the subsequent simulation of FLRT-binding enabled the researchers to identify the binding sites, where the three proteins interconnect. By introducing minimal changes, the scientists could interrupt these binding sites.
As a result, the migration behavior of the embryonic neurons changed in the brains of mice.
During brain development, embryonic neurons migrate to “their” brain area. As the investigations have now shown, the three proteins help to guide the cells to their destination.
“Surprisingly, this happens not by attraction, as in synapse formation, but by repulsion of the cells,” explains Rüdiger Klein from the Max Planck Institute of Neurobiology. “This function was completely new and unexpected,” adds Elena Seiradake from Oxford University.
Embryonic neurons often have only a cell body and short protrusions, called neurites. When Teneurin and FLRT on these structures bind to Latrophilin, the cells repel each other. As a result, the migrating cells partially lose their hold and progress more slowly.
Thus guided, the cells reach their target brain area at the right time, where they mature and form a long axon.
The interaction between three proteins shoves young nerve cells to their destination in the brain. Towards the end of brain development, however, the proteins hold together, enabling synapses to form. Image is credited to Falconieri Visuals, LLC and del Toro et al. in Cell, January 2020.
However, when on the surface of such an axon, Teneurin and FLRT no longer trigger a repulsive reaction upon the encounter with Latrophilin. Here and now, the proteins pull the cells together, induce the formation of synapses and ultimately lead to the assembly of networks of communicating neurons.
“The same proteins thus lead to completely different reactions – depending on their location on the cell,” summarizes Elena Seiradake the results.
“We now have ideal conditions to investigate further interactions of the proteins during brain development,” explains Rüdiger Klein.
In their previous studies, the researchers were able to show that FLRT influences both the migration behavior of young nerve cells and the formation of folds on the brain surface via interactions with its own binding partners.
“It will be exciting to see whether and how Teneurin and Latrophilin are involved in these interactions,” says Klein.
In brain, synaptic connections form neuronal communication networks, thereby constructing neural circuits. Synaptic connections are exquisitely specific and dynamic, but the underlying molecular mechanisms remain largely unexplored. In the hippocampus, Schaffer-collateral axons from the CA3 region form synapses on CA1 region pyramidal neurons exclusively on dendritic domains in the S. oriens and S. radiatum of these neurons.
In contrast, perforant-path axons from the entorhinal cortex form synapses on CA1 region pyramidal neurons exclusively on dendritic domains in the S. lacunosum-moleculare. How this synaptic input specificity is achieved, however, and what signaling mechanisms maintain the two classes of synapses, is unknown.
Synapse formation is thought to involve bidirectional signaling by trans-synaptic cell-adhesion molecules. Building on recent observations that the adhesion G-protein coupled receptor (GPCR) latrophilin-2 is essential for synapses in the S. lacunosum-moleculare of the CA1 region, we asked whether distinct latrophilins are localized to different dendritic domains of CA1 region neurons.
Moreover, latrophilins are known to form trans-cellular interactions with two classes of cell-adhesion molecules, teneurins and fibronectin leucine-rich-repeat transmembrane proteins (FLRTs).
Thus we hypothesized that latrophilins may act in synapse formation via trans-synaptic interactions with these adhesion molecules as ligands, and that such interactions may contribute to the specificity of synapse formation.
We produced genetic manipulations that allow monitoring the localization of endogenous latrophilin-2 and latrophilin-3 in vivo and that enable their conditional deletion. Using these manipulations, we found that latrophilin-2 and latrophilin-3 were specifically localized to postsynaptic spines in non-overlapping dendritic domains of CA1 region pyramidal neurons. Latrophilin-2 was targeted only to excitatory synapses in the S. lacunosum-moleculare, whereas latrophilin-3 was targeted only to excitatory synapses in the S. oriens and S. radiatum, corresponding to distinct presynaptic inputs onto CA1 region pyramidal neurons. Deletion of latrophilin-3 selectively decreased Schaffer-collateral synapses in the S. radiatum and S. oriens, whereas deletion of latrophilin-2 selectively decreased entorhinal cortex-derived synapses in the S. lacunosum-moleculare of CA1 neurons.
In vivo rescue experiments with latrophilin-3 mutants that selectively lack binding to only FLRTs or only teneurins revealed that both binding activities were required for input-specific synapse formation, as monitored by electrophysiology and retrograde rabies tracing. Thus, coincident binding of both latrophilin-3 ligands was necessary for synapse formation.
Moreover, in in vitro synapse formation assays teneurin-2 or FLRT3 alone were unable to induce excitatory synapse formation, whereas together they potently did so. However, even in combination FLRT3 and teneurin-2 only induced excitatory synapses when teneurin-2 was expressed as a splice variant that is competent to interact with latrophilins, indicating that simultaneous binding of both FLRT3 and teneurin-2 to latrophilins was necessary to induce synapse formation.
We suggest that latrophilin-2 and latrophilin-3 are postsynaptic adhesion GPCRs that are targeted in CA1 pyramidal neurons to non-overlapping dendritic domains, where they promote excitatory synapse formation by specific and distinct presynaptic inputs. The function of latrophilin-3 in synapse formation required simultaneous binding of two unrelated presynaptic ligands, FLRTs and teneurins, suggesting a coincidence signaling mechanism that could account for the specificity of synaptic connections.
Postsynaptic latrophilin-2 and latrophilin-3 mediate synapse specificity by simultaneous binding to presynaptic FLRTs and teneurins. Latrophilin-2 and latrophilin-3 are exclusively localized to dendritic domains of CA1 pyramidal neurons in the S. lacunosum-moleculare or the S. oriens and S. radiatum, respectively. In these locations, latrophilins are essential for synapse formation by simultaneously interacting with two different presynaptic cell-adhesion molecules, FLRTs and teneurins.
Max Planck Institute