Evaluating a cell type-specific mechanism of glutamatergic synapse function and organization - Project Summary/Abstract Impaired inhibitory signaling underlies the development of various neuropsychiatric disorders including anxiety, epilepsy, autism, and schizophrenia. Inhibition is mediated by interneurons (INs), a small but diverse population of cells that coordinates network activity of principal neuron underlying cognition. Glutamatergic excitation of INs determines subsequent firing and control of downstream circuits, and glutamatergic synapses received by INs have unique basal transmission properties and exhibit distinct synaptic plasticity compared to excitatory neurons. These differences in functionality are likely due to cell-type specific differences in postsynaptic density (PSD) composition and maintenance mechanisms. For example, recently identified postsynaptic proteins such as Btbd11 which are expressed exclusively in glutamatergic IN-PSD could underly the unique synaptic properties seen in INs. Growing evidence supports the idea that the PSD is organized through liquid-liquid phase separation (LLPS), the process of maintaining protein-dense, membrane-less organelles. Btbd11 has been shown to i) regulate synaptic function in INs and ii) undergo LLPS with key glutamatergic scaffolding proteins. However, whether Btbd11’s regulation of glutamatergic synapses requires LLPS is not yet known, and the extent to which Btbd11 mediates synaptic composition has not been explored. To investigate how the protein composition of glutamatergic IN-PSD contributes to the unique function of INs and subsequent role in pathophysiology, this application will utilize molecular biology, proteomic analysis, live cellular imaging and electrophysiology to address the novel hypothesis that Btbd11 promotes glutamatergic IN-PSD function through stabilizing interactions with Psd-95 and associated protein complexes via LLPS. In Aim 1, we will examine how Btbd11 ablation alters the IN-PSD proteome and levels of glutamatergic signaling molecules to determine a role in synaptic organization. In Aim 2, we will investigate how Btbd11’s phase separation properties and phospho-regulation are crucial for synaptic function and PSD protein stability through Btbd11 knockout and rescue experiments. Together, successful completion of the proposed project will provide insight into a cell-type specific mechanism of stability and function of an essential but understudied synapse, while further revealing the role of phase separation at the synapse. Through this proposal, I will be thoroughly trained in molecular biology, electrophysiology, live cellular imaging, and bioinformatics. In addition, mentorship from Dr. Bygrave and co-sponsor Dr. Maguire will provide an environment with exceptional guidance and rigorous training, access to all necessary equipment, and opportunities for networking, mentorship, and career development activities. Collectively, this proposal will provide me with the scientific training and growth necessary for establishing the foundations for a future successful career as an independent neuroscience researcher.
