Molecular mechanism of the synapse organizer C1QL3

ABSTRACT Synaptic adhesion molecules (SAMs) control the structural and functional properties of synapses and thereby shape neural circuit development and function. Genetic variants of SAMs are linked to various neurological disorders, but the mechanisms by which they exert such pathogenic effects remain elusive. The central goal of this research plan is to determine the mechanism by which the secreted synaptic protein C1QL3 promotes excitatory synaptogenesis. It was previously shown that C1QL3-deficient mice exhibited fewer excitatory synapses and multiple behavioral abnormalities. In preliminary work, we have demonstrated that C1QL3 mediates the formation of a cell-cell adhesion complex via interactions with both neuronal pentraxins (NPTX1/R) and adhesion G protein-coupled receptor B3 (ADGRB3). Furthermore, in a screen for novel C1QL3 binding partners, we identified a candidate matricellular protein involved in the regulation of matrix metalloproteinase (MMP) activity and showed that its overexpression increases excitatory synapse density in cultured neurons. These results suggest an MMP-mediated regulatory mechanism for the action of the C1QL3 adhesion complex. Our overarching hypothesis is that C1QL3 and MMPs co-modulate synaptic plasticity by regulating a novel cell- cell adhesion complex at excitatory synapses. To characterize C1QL3 interactions with its binding partners and to advance our understanding of these interactions in governing C1QL3 functions, we have developed the following specific aims. 1. To elucidate the structural basis for the transsynaptic interactions of C1QL3 with both ADGRB3 and NPTX1/R using NMR spectroscopy, mutagenesis, and cell-based assays. We hypothesize that such interactions promote synapse formation and maintenance. We have obtained a 1H-15N HSQC spectrum of the binding domain of C1QL3 and will carry out chemical shift perturbation (CSP) titrations with the corresponding interacting domains of NPTX1 and ADGRB3. We will identify each binding interface, and characterize mutations that will weaken these interactions. Mutant C1QL3 proteins will be tested in vitro for their ability to promote both the formation of the adhesion complex and cell-cell adhesion. We will look for correlations between the structural and physiological effects of the mutations. 2. To elucidate the mechanism by which MMPs regulates C1QL3- mediated synapse homeostasis. We hypothesize C1QL3 interacts with a novel protein that interferes with the proteolytic cleavage of the C1QL3 adhesion complex by MMPs. CSPs will be used to identify specific C1QL3 residues involved in interactions with this protein. We will investigate, as in Aim 1, the effects of mutations on these interactions in cellular contexts. This exploratory proposal will provide insights into the assembly and regulation of a novel transsynaptic adhesion complex and enhance our understanding of how this complex controls synapse structure and function. 1