A systematic structure-function characterization of a human mutation in Neurexin-3α reveals an extracellular modulatory sequence that stabilizes neuroligin-1 binding to enhance the postsynaptic properties of excitatory synapses

Abstract

α-neurexins are essential and highly expressed presynaptic cell-adhesion molecules that are frequently linked to neuropsychiatric and neurodevelopmental disorders. Despite their importance, how the elaborate extracellular sequences of α-neurexins contribute to synapse function is poorly understood. We recently characterized the presynaptic gain-of-function phenotype caused by a missense mutation in an evolutionarily conserved extracellular sequence of neurexin-3α (A687T) that we identified in a patient diagnosed with profound intellectual disability and epilepsy. The striking A687T gain-of-function mutation on neurexin-3α prompted us to systematically test using mutants whether the presynaptic gain-of-function phenotype is a consequence of the addition of side-chain bulk (i.e., A687V) or polar/hydrophilic properties (i.e., A687S). We used multidisciplinary approaches in mixed-sex primary hippocampal cultures to assess the impact of the neurexin-3α^A687 residue on synapse morphology, function and ligand binding. Unexpectedly, neither A687V nor A687S recapitulated the neurexin-3α A687T phenotype. Instead, distinct from A687T, molecular replacement with A687S significantly enhanced postsynaptic properties exclusively at excitatory synapses and selectively increased binding to neuroligin-1 and neuroligin-3 without changing binding to neuroligin-2 or LRRTM2. Importantly, we provide the first experimental evidence supporting the notion that the position A687 of neurexin-3α and the N-terminal sequences of neuroligins may contribute to the stability of α-neurexin-neuroligin-1 trans-synaptic interactions and that these interactions may specifically regulate the postsynaptic strength of excitatory synapses.Significance Statement Although neurexins were discovered over 30 years ago, our understanding of how the complex extracellular sequences unique to α-neurexins participate in synapse function remains incomplete. We leveraged a previously studied human missense mutation, located in a conserved extracellular region of neurexin-3α and linked to profound intellectual disability and epilepsy, to systematically assess the tolerance of neurexin-3α function to mutations within this region. Using molecular replacement, we assessed how single amino acid substitutions in this extracellular region alters synapse morphology, presynaptic calcium dynamics, and synaptic transmission. We reveal that multiple neurexin ligands unexpectedly use this region to modulate trans-synaptic binding and that different amino acid substitutions in place of the disease mutation result in dramatically different changes to synaptic transmission.