The molecular logic of trans-synaptic signaling : insights from the olfactory bulb

The information processing by the brain depends on the immense yet specific synaptic connections among neurons. The formation and functional properties of the synapses are regulated by trans-synaptic signaling molecules that are often cell adhesion molecules. To illustrate the importance of trans-synaptic signaling, I first identified a molecularly and anatomically defined synaptic circuit essential for a moue behavior called social transmission of food preference (STFP) in Chapter 1. C1q like 3 (C1ql3) is a neuronal secreted protein with implications in excitatory synaptogenesis and is a ligand for cell-adhesion G protein-coupled receptor (GPCR) Brain Angiogenesis Inhibitor 3 (Bai3). In Chapter 1, I showed that the ligand-receptor pair C1ql3-Bai3 is essential for the synaptic transmission of the feedback projections (i.e. centrifugal projections) from a central olfactory region - anterior olfactory nucleus (AON) to the olfactory bulb (OB). Deletion of presynaptic C1ql3 from AON or postsynaptic Bai3 from the OB impaired specifically the acquisition but not recall of STFP. Hence, Chapter 1 illustrates the importance of trans-synaptic signaling in regulating synaptic properties and thus animal behaviors. Admittedly, tremendous progress has been made in the past decades on the various synaptic adhesion molecules (SAMs) in the cell biology of synapse formation, as well as their implications in animal behaviors and psychiatric disorders. However, we still lack a conceptual framework to understand the molecular logic of trans-synaptic signaling by SAMs. Possibly due to the shared architecture of presynaptic release machinery and the diversity of postsynaptic components, presynaptic SAMs often have the potential to interact with a diverse set of postsynaptic ligands. Notably, neurexins are presynaptic SAMs with the potential to interact with postsynaptic transmembrane ligands such as neuroligins, LRRTMs, dystroglycans, as well as neuronal secreted molecules like cerebellins. Hence, one major conceptual challenge is how presynaptic SAMs engage in the proper postsynaptic ligands despite their binding promiscuity. I will focus on neurexins as an example to tackle this conceptual challenge in Chapters 2 & 3, because of the well-characterized biochemical properties of neurexins. Neurexins are encoded by three genes Nrxn1/2/3 in the mammalian genome, and each gene can be transcribed from two distinct promoters to form a longer α- and a shorter β-isoform. Nrxn1 can also be transcribed from a third promoter to produce an even shorter γ-isoform. Moreover, Nrxns are highly alternatively spliced with six different splicing sites (SS1-6), all on the extracellular domains. The diversity of neurexin isoforms and splicing variants not only underlies their binding promiscuity, but also determines the binding specificity of neurexins. For example, neurexins must include the SS4 exon to be able to interact with cerebellins. In contrast, neurexins can only interact with LRRTMs if they do not contain SS4. Because of the interaction specificity encoded by different splicing sites, the alternative splicing of neurexins has always been thought as the code for neurexin signaling. In this dissertation, I aim at addressing this fundamental question by using the reciprocal synapse in the olfactory bulb (OB) as a model synapse, in analogy to the idea of choosing a model organism to tackle a specific biological question. The reciprocal synapse in the OB is formed between two neighboring dendrites from mitral/tufted cells and granule cells. The mitral/tufted cells are the only projection neurons in the olfactory bulb, while the granule cells are the local inhibitory neurons. In the reciprocal synapse, the mitral/tufted cells form excitatory synapses onto granule cells, while the granule cells form inhibitory synapses onto mitral/tufted cells. These two antiparallel synapses exist right next to each other without any physical compartmentalization, sharing pre- and post-synaptic compartments. In Chapter 2, I used the granule cell-to-mitral cell (GCMC) synapses as a model because previous work established the role of Nrxn3α in regulating the presynaptic release from granule cells at these synapses. Following up with the work done in the olfactory bulb culture by Justin H. Trotter and Peng Zhou, I first designed CRISPR strategies to manipulate the level of dystroglycan, a postsynaptic ligand for α-neurexins as long as the neurexins do not contain splice site 2 (SS2), and showed its importance in regulating both presynaptic release and postsynaptic GABA receptor response at GCMC synapses. Moreover, by molecular replacement of endogenous Nrxn3 in the OB, I further showed that SS2-lacking Nrxn3α forms the functional trans-synaptic complex with dystroglycan in regulating the presynaptic release from granule cells. Hence, Chapter 2 shows that the alternative splicing of neurexins permits protein-protein interaction to exert trans-synaptic signaling. In Chapter 3, I utilized the unique structure of the reciprocal synapse to address the question of molecular self-avoidance in synaptic neurexin complexes. Since both mitral/tufted cells and granule cells express neurexins, the neurexins on either side can interact with their ligands in both cis and trans configurations. Due to their proximity, neurexins at reciprocal synapses will preferentially interact with their ligands in cis, thus interfering with the proper trans interactions. Hence, a molecular self-avoidance mechanism to prevent cis interaction is necessary for the parallel operation of the two antiparallel neurexin trans-synaptic signaling pathways. I first showed that both neurexin ligands Cbln1 and neuroligins from mitral cells mediate the postsynaptic GABA response at the reciprocal synapse. Moreover, mitral cells express neurexins predominantly containing SS4, allowing them to interact with Cbln1, and conversion of the SS4 from inclusion to exclusion for all neurexins in mitral cells recapitulates the same deficit caused by Cbln1 and neuroligins deletion. At last, by using in vitro modeling through the cell aggregation assay and in vivo genetic epistasis, I showed that Cbln1 together with biased SS4+ splicing of neurexins in mitral cells is the self-avoidance mechanism in the synaptic neurexin complexes. Therefore, Chapter 3 provides an example where the alternative splicing of neurexins permits protein-protein interactions to avoid an unwanted cis ligand-receptor interaction. Viewed together, this dissertation first illustrates the importance of trans-synaptic signaling in animal behaviors by studying the role of C1ql3 and Bai3 in centrifugal projections as well as STFP. The last two chapters of the dissertation focus on the presynaptic SAM neurexins and utilize the previous knowledge and unique structure of the reciprocal synapse to illustrate how the alternative splicing of neurexins dictates the specificity of ligand-receptor interactions to allow proper trans-synaptic signaling to occur while mediating the self-avoidance in the synaptic neurexin complexes. By no means this dissertation fully reveal the molecular logic of trans-synaptic signaling by neurexins, letting alone other SAMs in general. However, by taking advantage of the reciprocal synapse as a model synapse, this dissertation not only identifies a novel functional trans-synaptic complex in regulating presynaptic release, but also shows how multiple molecular machineries operate in parallel within the trans-synaptic signaling network with promiscuous protein-protein interactions

Advisor: Südhof Thomas C.
Kaltschmidt Julia
Luo Liqun
Wang Sui