Control of subsynaptic domain organization and nanocolumn alignment by neurexin-3

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Project Summary/Abstract Efficient synaptic communication between neurons requires precise transcellular alignment of the presynaptic terminal with the postsynaptic specialization. The sites of neurotransmitter release and detection are not random; instead the active zone release machinery is locally enriched and precisely aligned transsynaptically with locally enriched postsynaptic scaffolds and receptors. Recently, super-resolution microscopy approaches have revealed that many proteins critical for synaptic transmission are not uniformly distributed but highly enriched in subsynaptic domains (SSDs). These SSDs are aligned to form transsynaptic nanocolumns that are commonly thought to regulate the efficacy of synaptic transmission. An outstanding question is: how are SSDs regulated, localized and aligned into transsynaptic nanocolumns? Transsynaptic cell adhesion molecules have been proposed to control the nanoscale organization of synapses because they span the synaptic cleft and are capable of participating in bidirectional signaling via intracellular and extracellular sequences. Underscoring the importance of adhesion molecules, mutations in genes that encode these proteins are commonly linked to mental health and substance use disorders. While adhesion molecules studied thus far cluster in SSDs in or around the synapse, the endogenous manipulation of presynaptic adhesion proteins has yet to reveal an instructive role for these molecules in controlling transsynaptic SSD properties. Using 3D dSTORM superresolution imaging, we have identified neurexin-3 (Nrxn3) as the first neurexin and first presynaptic adhesion molecule necessary for the nanoscale organization of excitatory synapses. Conditional ablation of neurexin-3 in primary hippocampal cultures significantly reduced the synaptic density, volume and transsynaptic alignment of active zone and postsynaptic SSDs. We hypothesize that distinct Nrxn3 signaling sequences govern SSD properties and nanocolumn alignment and that neurexin-3 forms presynaptic SSDs directly aligned with postsynaptic density SSDs. This proposal builds on our preliminary and published data that neurexin-3 is necessary for subsynaptic organization and critical for AMPAR-mediated synaptic transmission. We will utilize 3D dSTORM imaging, molecular structure/function approaches and mouse genetics to test our hypothesis in two specific aims. First, we will directly examine the role of neurexin-3 on AMPA-receptor SSDs at excitatory synapses and systematically perform structure/function experiments to determine the intracellular and extracellular signaling sequences of neurexin-3 required for nanoscale organization and SSD alignment. Second, we will investigate the subsynaptic localization and transsynaptic alignment of endogenous neurexin-3. Together, our findings will provide critical insight into how neurexin-3 signaling governs subsynaptic architecture and how dysfunction of neurexin-3 can contribute to the etiologies that underlie neuropsychiatric, neurodevelopmental and substance use disorders.