Characterizing the Trafficking and Function of Nedd4-1 in the Neuron

The purpose of this dissertation is to bring a greater understanding of the trafficking and function of the ubiquitin E3 ligase, neuronal expressed downregulated in development protein 4-1 (Nedd4-1), in the neuron. Neuronal cell health depends on the delicate regulation of the various protein abundances across the neuron through protein homeostasis (proteostasis). Proteostasis consists of three major parts: protein synthesis, maintenance, and degradation. Ubiquitination by the E3 ligase plays an important part in proteostasis by targeting proteins for degradation. In this dissertation, I focus on studying how Nedd4-1’s trafficking and protein interactions are tightly controlled by synaptic activity. These studies help better elucidate Nedd4-1’s normal function in neurons and implications in neurological disease. In Chapter II, I present a comprehensive review of Nedd4-1’s described role in the central nervous system (CNS) in health and disease. This provides context for the experimental questions posed throughout this thesis. In Chapter III, we sought to understand rules regulating Nedd4-1 trafficking in neurons. Because cellular compartments differ greatly, subcellular localization of proteins is often finely regulated and intimately tied to their function. We created a GFP-tagged Nedd4-1 expression construct for use in live time-lapse imaging in hippocampal neurons to study the trafficking dynamics of Nedd4-1. First, we validated the utility of GFP-Nedd4-1 in live imaging. Next, we show that α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor and N-methyl-D-aspartate (NMDA) receptor activation drive distinct trafficking dynamics of GFP-Nedd4-1 in neurons. Together these data suggest Nedd4-1’s trafficking is subject to fine and distinct activity-dependent regulation in neurons. In Chapter IV, we further explored Nedd4-1’s function in neurons by using APEX2 mediated proximity labeling to characterize Nedd4-1’s interactome. We created and validated the utility of APEX2-HA-Nedd4-1 expression and proximity labeling in primary neuronal cultures. We show that in untreated neuronal cultures, APEX2-Nedd4-1 had a significant association with spliceosome complex components and mRNA processing pathways. In contrast, NMDAR activation significantly increased APEX2-Nedd4-1 interaction with synaptic proteins. Using co-localization with postsynaptic marker PSD-95 and pharmacological inhibition of Nedd4-1 we show that NMDARs recruit Nedd4-1 to the synapse to possibly participate in regulation of GluA1 containing AMPARs there