NSF-BSF: Uncovering the specific mechanisms of spine and axonal pruning mediated by Semaphorin-Plexin signaling

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Brain circuits wire themselves by first forming too many connections between nerve cells (neurons), followed by a precise refinement of these excess connections to produce fully functional brain networks. Connections are refined by pruning the neuronal structures that cells connect with, called dendritic spines (on the receiving end) and axonal terminals (on the sending end). Proper pruning of neuronal connections is essential for the proper organization and function of the nervous system; disrupted pruning results in dysfunctional brain connections and abnormal behavior in organisms. The ways in which the developmental removal of connections are regulated is one of the big unsolved mysteries of the brain. The aim of this joint project is to uncover how a family of molecules called Semaphorins instructs the elimination of excess connections in a brain region important for memory formation and spatial orientation/recognition behavior in both rodents and humans (the hippocampus). To address the research objectives, the project will use isolated mouse neurons grown in an incubator, as well as intact neurons inside mouse brains that have been genetically modified in specific ways to examine the role that Semaphorins play in axonal and dendritic pruning during brain development. In addition, the project will introduce large numbers of undergraduate students to cutting-edge 3-dimensional brain image analysis techniques, and expand a successful science outreach program for high school students conducted at a local school with a 98% minority enrollment (and where 87% of the students are economically disadvantaged), as well as providing in-depth research training and participation to undergraduate students. This study will provide new mechanistic insights into how neurons establish their normal connections, and will provide answers to the long-standing question of how specific molecules regulate the complex process of neuronal circuit refinement. During brain development, guidance cues control the complex pruning processes that dendritic spines (postsynaptic) and axonal terminals (presynaptic) undergo, mainly through the activation of receptors on the responding neurons. The specific intracellular signaling mechanisms underlying how guidance cues induce these responses are poorly understood. Interestingly, the same Semaphorin/Plexin-signaling system (ligand-receptor pair) appears to operate on both the postsynaptic and presynaptic side of mouse hippocampal granule cells to induce spine and axonal pruning, respectively. In this project, novel genetically-modified mouse lines (generated using CRISPR/cas9 methodology) will be combined with a variety of in-vitro and in-vivo approaches to ask how the Semaphorin/Plexin-signaling pathway(s) regulate synaptic pruning at three different levels. The specific aims will address: 1) the identity of the ligand/receptor pair(s) responsible for spine versus axon pruning, 2) the role of specific cytoplasmic motifs within the Plexin receptors and, 3) the novel Plexin downstream signaling elements involved with each process. This study brings together expertise in molecular, cellular biology, biochemistry, and mouse genetics, in a synergistic and collaborative effort to investigate the specific mechanisms that control dendritic spine and axonal pruning in the developing organism. The anticipated findings will provide a new level of understanding about the molecular mechanisms that govern the wiring of the mammalian brain.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.