Teneurins are large cell-surface receptors involved in axon guidance. Teneurin-2 (also known as latrophilin-1-associated synaptic surface organizer (Lasso)) interacts across the synaptic cleft with presynaptic latrophilin-1, an adhesion G-protein-coupled receptor that participates in regulating neurotransmitter release. Lasso-latrophilin-1 interaction mediates synapse formation and calcium signaling, highlighting the important role of this trans-synaptic receptor pair. However, Lasso is thought to be proteolytically cleaved within its ectodomain and released into the medium, making it unclear whether it acts as a proper cell-surface receptor or a soluble protein. We demonstrate here that during its intracellular processing Lasso is constitutively cleaved at a furin site within its ectodomain. The cleaved fragment, which encompasses almost the entire ectodomain of Lasso, is potentially soluble; however, it remains anchored on the cell surface via its non-covalent interaction with the transmembrane fragment of Lasso. Lasso is also constitutively cleaved within the intracellular domain (ICD). Finally, Lasso can be further proteolytically cleaved within the transmembrane domain. The third cleavage is regulated and releases the entire ectodomain of Lasso into the medium. The released ectodomain of Lasso retains its functional properties and binds latrophilin-1 expressed on other cells; this binding stimulates intracellular Ca(2+) signaling in the target cells. Thus, Lasso not only serves as a bona fide cell-surface receptor, but also as a partially released target-derived signaling factor.

Synaptogenesis requires formation of trans-synaptic complexes between neuronal cell-adhesion receptors. Heterophilic receptor pairs, such as neurexin Ibeta and neuroligin, can mediate distinct intracellular signals and form different cytoplasmic scaffolds in the pre- and post-synaptic neuron, and may be particularly important for synaptogenesis. However, the functions of neurexin and neuroligin depend on their distribution in the synapse. Neuroligin has been experimentally assigned to the post-synaptic membrane, while the localization of neurexin remains unclear. To study the subcellular distribution of neurexin Ibeta and neuroligin in mature cerebrocortical synapses, we have developed a novel method for the physical separation of junctional membranes and their direct analysis by western blotting. Using urea and dithiothreitol, we disrupted trans-synaptic protein links, without dissolving the lipid phase, and fractionated the pre- and post-synaptic membranes. The purity of these fractions was validated by electron microscopy and western blotting using multiple synaptic markers. A quantitative analysis has confirmed that neuroligin is localized strictly in the post-synaptic membrane. We have also demonstrated that neurexin Ibeta is largely (96%) pre-synaptic. Thus, neurexin Ibeta and neuroligin normally form trans-synaptic complexes and can transduce bidirectional signals.

The GluR3 subunit of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) has been identified as a target for autoantibodies (Aabs) in autoimmune encephalopathy and other diseases. Recent studies have proposed mechanisms by which these Aabs act, but their exact role in neuronal excitability is yet to be established. Patient Aabs have been shown to bind to specific regions within the GluR3 subunit. GLUR3B peptides were designed based on described (ELISA) immunogenic epitopes for Aabs and an immunisation strategy was used to generate novel anti-AMPAR Aabs. Target-specific binding and specificity of affinity-purified anti-AMPAR Aabs was confirmed using enzyme-linked immunosorbent assay, immunocytochemistry and Western blot. Functional anti-AMPAR Aab effects were determined on excitatory postsynaptic currents (EPSCs) from primary hippocampal neurons using whole-cell patch-clamp electrophysiology. Acute (10 or 30 min) or longer-term (24 h) application of anti-AMPAR Aabs caused a significant reduction in the mean frequency of spontaneous and miniature EPSCs in hippocampal neurons. Our data demonstrate that anti-AMPAR Aabs targeting peptides linked to auto-immune diseases mediate inhibitory effects on neuronal excitability at the synaptic level, such effects may lead to disruption of the excitatory/inhibitory balance at a network level.

Inflammatory skin conditions are increasingly recognised as being associated with systemic inflammation. The mechanisms connecting the cutaneous and systemic disease are not well understood. CD1a is a virtually monomorphic major histocompatibility complex (MHC) class I-like molecule, highly expressed by skin and mucosal Langerhans cells, and presents lipid antigens to T-cells. Here we show an important role for CD1a in linking cutaneous and systemic inflammation in two experimental disease models. In human CD1a transgenic mice, the toll-like receptor (TLR)7 agonist imiquimod induces more pronounced splenomegaly, expansion of the peripheral blood and spleen T cell compartments, and enhanced neutrophil and eosinophil responses compared to the wild-type, accompanied by elevated skin and plasma cytokine levels, including IL-23, IL-1α, IL-1β, MCP-1 and IL-17A. Similar systemic escalation is shown in MC903-induced skin inflammation. The exacerbated inflammation could be counter-acted by CD1a-blocking antibodies, developed and screened in our laboratories. The beneficial effect is epitope dependent, and we further characterise the five best-performing antibodies for their capacity to modulate CD1a-expressing cells and ameliorate CD1a-dependent systemic inflammatory responses. In summary, we show that a therapeutically targetable CD1a-dependent pathway may play a role in the systemic spread of cutaneous inflammation.

A presynaptic adhesion G-protein-coupled receptor, latrophilin-1, and a postsynaptic transmembrane protein, Lasso/teneurin-2, are implicated in trans-synaptic interaction that contributes to synapse formation. Surprisingly, during neuronal development, a substantial proportion of Lasso is released into the intercellular space by regulated proteolysis, potentially precluding its function in synaptogenesis. We found that released Lasso binds to cell-surface latrophilin-1 on axonal growth cones. Using microfluidic devices to create stable gradients of soluble Lasso, we show that it induces axonal attraction, without increasing neurite outgrowth. Using latrophilin-1 knockout in mice, we demonstrate that latrophilin-1 is required for this effect. After binding latrophilin-1, Lasso causes downstream signaling, which leads to an increase in cytosolic calcium and enhanced exocytosis, processes that are known to mediate growth cone steering. These findings reveal a novel mechanism of axonal pathfinding, whereby latrophilin-1 and Lasso mediate both short-range interaction that supports synaptogenesis, and long-range signaling that induces axonal attraction.