The phase separation of neuronal pentraxins: from synapse formation to plasticity.

Although the synapse was discovered more than a century ago, the fundamental mechanisms mediating the processes of synaptogenesis and synaptic plasticity remain incompletely understood. Understanding how synapses are properly formed and regulated during plasticity is crucial given that disruptions in these processes are closely associated with a broad array of neurological and psychiatric disorders. In this proposal, I introduce a novel avenue for exploring synapse formation and synaptic plasticity, focusing on my unexpected observation that neuronal pentraxins—the synaptic cell adhesion molecules expressed by neurons to regulate synaptic strength—undergo liquid-liquid phase separation (LLPS). This phase separation and subsequent clustering of AMPA receptors by all three neuronal pentraxins (Nptx1, Nptx2, and Nptxr) highlight a potentially groundbreaking
mechanism in synaptic organization. My central hypothesis is that phase separation mediated by neuronal pentraxin-based cell adhesions coordinates synaptic structural changes and AMPA receptor clustering during both synaptogenesis and long-term potentiation (LTP). To test this hypothesis, I propose an integrated approach combining mouse genetics, synapse super-resolution imaging, biochemical reconstitution, structural biology, and proximity labeling. This multifaceted strategy will enable me to determine Nptxr's role in synapse assembly using Nptxr cKO mice (Aim1), elucidate the mechanism behind Nptxr's phase separation and its impact on synapse assembly and AMPA receptor clustering in synaptogenesis, by using structural biology techniques (Aim 2), and examine how Nptx2's LLPS, triggered by synaptic activity, influences synapse formation and maturation during
LTP utilizing Nptx2 cKO mice (Aim 3). Results from these experiments promise to deepen our understanding of how protein phase separation at synaptic cleft regulates synapse assembly, thus contributing not only insight into how synapses are formed and function, but also into how LTP is induced and expressed. More importantly, this research will also provide a basic understanding of neuronal pentraxin’s roles in neurodegenerative and neuropsychiatric disorders such as Alzheimer's disease, Parkinson's disease, autism, and schizophrenia, to which neuronal pentraxins have been linked genetically. These aims will be supported by an exceptional mentoring team of Drs. Thomas Südhof and Axel Brunger, with advisory contributions from Drs. Wah Chiu, Ivan Soltesz, Xiaowei Zhuang, Alice Ting, and Le Cong, within the stimulating research environment of Stanford
University. This award will bolster my career development through personalized training in molecular
neuroscience, cryo-EM structural biology, and synaptic plasticity. Then comprehensive training regimen is designed to ensure the successful completion of the proposed research and facilitate my transition to an independent role focused on exploring synapse functions mediated by the phase separation of synaptic proteins.

Grant Number: 1K99NS140628-01A1
NIH Institute/Center: NIH
Principal Investigator: Xudong Chen