Establishing New Genetic and Pharmacological Based Models to Investigate the Role of Nedd4-1 in AMPA Receptor Trafficking

The purpose of this dissertation research is to advance our understanding of the molecular mechanisms underlying synaptic plasticity and neuronal communication. Neurotransmitter receptors mediate signal transduction between neurons, and signaling mechanisms are dependent on the identity and quantity of receptors expressed on the surface of the neurons. Surface receptor expression – as a result of e.g. synthesis, trafficking, removal – must therefore be carefully regulated to maintain proper communication and thereby overall function and health. These receptors undergo protein modifications that choreograph their delivery, stabilization, or removal from the membrane surface. One such modifying enzyme is Neural precursor cell-expressed, developmentally down-regulated protein 4-1 (Nedd4-1), an E3 ubiquitin ligase, which has been shown to modify the GluA1 subunit of AMPA receptors with a ubiquitin molecule. While the implications that Nedd4-1 is involved in AMPAR-dependent forms of plasticity remain unambiguous, we aim to assess which of many forms of plasticity Nedd4-1 may be involved in and thus its role in the regulation of synaptic plasticity. In the following dissertation, Chapter II describes our efforts in characterizing a neuronal cell culture model. In this chapter, I specifically demonstrate that AAV-cre robustly abolishes Nedd4-1 protein in cultures derived from Nedd4-1 floxed mice. Importantly, unlike the original embryonically lethal in vivo knockout models or in vitro cultures with significant dendritic abnormalities, we find that in our neuronal cultures, knockout cells are morphologically healthy and amenable to standard biochemical, imaging, and physiology experiments, greatly facilitating our capacity to interrogate Nedd4-1 function in AMPAR trafficking and in health and disease. Finally, since Nedd4-1 is known to participate in the molecular mechanisms underlying synaptic plasticity, we aimed to delineate the specific forms of receptor internalization Nedd4-1 may be involved in. Chapter III provides a brief validation of a small molecule inhibitor of Nedd4-1, while Chapter IV expounds on our work to study two forms of plasticity paradigms. We found evidence that Nedd4-1 may be involved in AMPAR internalization following glycine-induced long-term potentiation, but not in NMDA-induced chemical long-term depression. These results help refine our understanding of Nedd4-1 function in neurons and point to new directions for future investigations