Development of antibodies for the detection of conformational changes in PSD-95 that regulate synaptic plasticity

Abstract Local protein phosphorylation is essential for synaptic plasticity, with proline-directed kinases such as GSK3β and CDK5 playing key roles in regulating synaptic modifications critical to long-term memory formation. Notably, phosphorylation of postsynaptic density protein 95 (PSD-95) by GSK3β is rapidly induced upon NMDAR-dependent long-term depression (NMDAR-LTD), destabilizing PSD-95 from the synapse and promoting its exit from postsynaptic spines. Proline- directed phosphorylation—a unique form of protein phosphorylation—can induce conformational changes in protein targets via cis-trans isomerization of the prolines following phosphorylated residues. Although the impact of this regulatory mechanism is well-characterized in cancer, its function in synaptic plasticity is largely unexplored. In this project, we aim to develop conformation- specific antibodies to detect cis and trans isomers of phospho-T19 in PSD-95, addressing a fundamental gap in our understanding of excitatory synapse biology. Specifically, we will investigate whether phosphorylation at serine/threonine-proline bonds in PSD-95 drives conformational changes that affect its biological function in synaptic plasticity. Our recent findings suggest that phosphorylation at T19 significantly alters the conformation of PSD-95’s N-terminus, affecting synaptic stability. The conformation-specific antibodies to be generated in this study will provide novel tools to directly monitor cis-trans isomerization states of PSD-95, enabling us to assess their role in synaptic stability and excitatory synaptic function in future studies. These experiments will offer unprecedented insights into the role of proline-directed phosphorylation and cis-trans isomerization in synaptic plasticity, advancing our broader understanding of regulatory mechanisms in excitatory synapses.