The complex neural processes of information encoding, storage, and retrieval are enabled by precise and efficient regulation of synaptic strength. Investigation into mechanisms of synaptic transmission will inform not only how we think about neural circuit functions, but also how we can better treat neurobiological diseases and disorders at a fundamental level. Our lab recently discovered a novel element of subsynaptic structure by which receptor activation may be modulated independent of conventional mechanisms. Proteins that establish presynaptic sites of neurotransmitter exocytosis are tightly aligned across the synapse with postsynaptic nanoclusters of receptors. This ?nanocolumn? of trans-synaptic structure is expected to impact synaptic efficacy by controlling the likelihood of receptor activation (Tang et al., 2016). However, despite much detailed examination of how receptors move in and around synapses, we almost completely lack understanding of the mechanisms that determine their positioning within the synapse and across from sites of release. Though many mechanisms may contribute to nanocolumn formation, a particularly attractive model is that synaptic cell adhesion molecules (CAMs) mediate alignment through high affinity trans-synaptic protein binding. My goal is to test this idea. However, distinguishing the ongoing roles of CAMs at synapses following their known roles in synaptogenesis is difficult. Synaptic CAMs undergo extensive splicing and include a large variety of proteins with similar functional domains, provoking widespread mechanistic compensation over the days following knockout or knockdown. To avoid these effects, I have been developing approaches to acutely perturb CAM trans-synaptic binding on the time scale of just minutes. My preliminary data adapts an approach originally developed by Peixoto et al. (2012) by inserting a protease cleavage site into the protein of interest, enabling acute and specific cleavage of desired protein domains. My design includes a knockdown-replacement strategy, permits independent tracking of the cleaved components, and can be expanded to target multiple proteins simultaneously. Here, I propose to apply my approach to test whether the synaptic CAM Leucine-Rich Repeat Transmembrane neuronal 2 (LRRTM2) mediates synaptic nanoalignment. LRRTM2 is a strong candidate to test first because it participates in trans-synaptic binding with key proteins (postsynaptic PSD-95 and presynaptic neurexin), it regulates synaptogenesis, and its knockdown results in decreased evoked EPSCs. Intriguingly, unlike most other CAMs, LRRTM2 also directly binds AMPARs within the postsynaptic density. With patch-clamp electrophysiology, super-resolution microscopy, and single-molecule tracking, I will use acute cleavage to test whether elimination of LRRTM2 extracellular interactions acutely disrupts trans- synaptic protein alignment, AMPA receptor mobility, and synaptic strength. These results will be the first test of an important new synaptic mechanism, and will provide key training establishing the basis for subsequent postdoctoral work.
Neurological and psychiatric disease frequently stem from a disruption of communication between cells of the nervous system. The experiments in this proposal are designed to improve our knowledge of how this trans- cellular communication is accomplished. By helping to identify the roles of particular molecules, the results here will provide insight that will influence possible design of treatment for disease.