My dissertation research focused on studying activity-dependent protein degradation in neurons primarily focusing on lysosomes, a degradative organelle. The role of lysosomes in regulating neuronal activity has been virtually unexplored, but their presence in dendrites was recently confirmed. My thesis work served to try to understand their role in dendrites, showing that lysosomes can be recruited to spines and their inhibition causes mEPSP frequency to decrease. I then expanded on this work to characterize their distribution throughout the entire dendrite, demonstrating that long term depression (LTD) and long term potentiation (LTP) cause lysosomes to shift proximally and distally respectively. This shift is associated with a decrease in surface GluA1 AMPA receptor subunits, suggesting that lysosomes may play a role in regulating the local intensity of LTP and LTD.In Chapter IV, I focus on AMPA receptor half-life by evaluating the effect of the phosphorylation state of serine 845 (s845). This phosphor-site has been shown to regulate internalization of GluA1. I found that phosphorylation of this site increases when neuronal activity is upregulated via bicuculline treatment, and that this phosphorylation changes over time, peaking between 4-8 hours. Inhibiting protein kinase A in conjunction with this treatment causes a synthesis-dependent degradation of GluA1. Chapter V describes the development of a method to purify lysosomes from neurons in preparation for proteomic analysis via mass spectrometry. This method was adapted to neurons from an existing method to purify lysosomes in dividing cells and shown to have the sensitivity to detect autophagy-induced changes in the lysosomal proteome.

The connections between neurons in the central nervous system are constantly changing in number and in strength. This process occurs through development and aging, but is also employed to enable learning and memory. Additionally, neurons must be able to constantly sense general activity patterns and make slow adjustments in a homeostatic manner in order for inputs to remain meaningful. One of the ways in which neurons can manage the control of their connections is through manipulations of proteins at synapses. This can be accomplished through control of protein synthesis, trafficking, and degradation. Glutamate receptors such as AMPA receptors as well as a number of postsynaptic scaffolding proteins are subject to a variety of posttranslational modifications and can be synthesized and degraded in an activity-dependent manner. Recently, it has become clear that multiple steps in degradation pathways can be affected by changes in synaptic activity, and that this regulation can have an immense impact on synaptic strength. However, the exact mechanisms through which degradation systems are regulated has largely remained a mystery. Here, we explore the regulation both of ubiquitination and proteasome-dependent degradation, uncovering novel ways in which synaptic activity interacts with these systems. First, we demonstrate that the activity-dependent ubiquitination of surface AMPA receptors is accomplished through synaptic recruitment of the ubiquitin ligase Nedd4-1, and show that this ligase is critical in homeostatic downscaling of synaptic strength. Additionally, we unveil a role for this ligase in the synaptic depression observed in models of Alzheimer’s disease, illustrating that regulation of Nedd4-1 can go awry. Next, we demonstrate that control of excitatory synapses can also be accomplished with the conjugation of the small ubiquitin-like molecule NEDD8, known to regulate the activity of a number of ubiquitin ligases. Finally, we explore the role of CaMKII-dependent phosphorylation of the Rpt6 subunit of 26S proteasomes in plasticity and learning. These studies add to our knowledge of how protein degradation can be regulated by activity and how this regulation can impact synaptic plasticity.

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.

The ADHD drug methylphenidate improves long-term memory, but it is still unclear whether this is a direct effect of the drug or due to improvements in executive function. Other ADHD pharmacological treatments, such as atomoxetine, improve executive control in terms of impulse control, working memory, and attention, but not long-term memory. Moreover, methylphenidate improves long-term memory in tasks with little attentional requirements, such as tone fear conditioning. This suggests that methylphenidate improves long-term memory through a mechanism that does not necessarily require improvements in executive function. This led us to hypothesize that methylphenidate directly enhances long-term memory. Frontal cortex is required for executive function, while the medial temporal lobe, including the hippocampus, is required for long-term memory formation. Methylphenidate binds targets in both the prefrontal cortex and hippocampus, but which of these is responsible for the improvements in long-term memory has yet to be determined. This study was undertaken to determine whether the frontal areas implicated in executive function are recruited in long-term memory enhancement by methylphenidate. Executive function was disrupted by lesioning prefrontal cortex in mice. Sham lesions were performed to account for the effects of surgery. Mice were then subjected to classical Pavlovian fear conditioning following administration of either methylphenidate or saline, and tested for contextual and tone memory one week later. In this study, both frontal lesions and methylphenidate failed to influence long-term memory, which may be due to increased stress from surgery that altered the optimal dose of methylphenidate. Further studies are required to elucidate the role of frontal cortex in methylphenidate-mediated memory enhancement.

Protein homeostasis in is critical to maintain cell health and viability. Protein homeostasis can be divided into two major categories: protein synthesis, and protein degradation. One of the major pathways of protein degradation is via the Ubiquitin Proteasome System (UPS), which, at its core, involves the proteasome; a multi-subunit cellular machine that unfolds and degrades proteins and breaks them down into small peptides to be utilized by the cell for further protein synthesis. Regulation of this machinery is vital to maintain protein homeostasis. We have discovered a posttranslational modification to the AAA ATPase Rpt6 subunit that modulates proteasome activity. Here, we have made mutants that abrogate or mimic this phosphorylation, in order to allow us to further uncover the regulatory mechanisms that govern protein breakdown and homeostasis in eukaryotic cells.

Chapter II investigates the basic cellular effects in the CNS of eliminating phosphorylation dynamics of Rpt6 in novel mouse models that either block Rpt6 phosphorylation or mimic it. Behavioral, electrophysiological, biochemical, and other techniques are utilized in investigating the effects of Rpt6 S120 phosphorylation in the mammalian brain.

In Chapter III, the loss of cocaine sensitization in Rpt6 S120D mice is explored. We find that treatment of cultured neurons with cocaine causes the phosphorylation and activation of CaMKIIα, and concomitant Rpt6 phosphorylation and subsequent increase in proteasome activity. This process is shown to be activated via monaminergic systems in neurons, specifically the inhibition of dopamine uptake. This process is abrogated when Rpt6 S120 phosphorylation is mimicked when mutated to aspartic acid, particularly in the nucleus accumbens and pre-frontal cortex, providing further insight into the pathogenesis of cocaine sensitization.

Chapter IV presents a study of the dynamics Rpt6 phosphorylation and cellular susceptibility to proteotoxic stress. Rpt6 S120 knock-in mutants of S. Cerevisiae are utilized to investigate how cells respond to various proteotoxic stressors. We find Rpt6 is phosphorylated in response to such stressors, and when phosphorylation is abrogated, cells are not able to respond to such stresses and become more susceptible to protein aggregation and cell death.

Changes in synaptic strength underlie the basis of learning and memory and are controlled, in part, by the insertion and removal of AMPA-type glutamate receptors. Once internalized, these receptors maybe recycled back to the plasma membrane by subunit-specific interactions with other proteins or by post-translational modifications such as phosphorylation. Ubiquitination, another post-translational modification, has recently emerged as a key signal that regulates the recycling and trafficking of glutamate receptors. However, the activity-dependent rules that govern internalization and degradation have not been fully elucidated.

This dissertation was designed in two parts; (1) to investigate the molecular specificity and activity-dependent rules of AMPAR ubiquitination via a deubiquinating enzyme, USP8 and (2) to determine how the lysosome, an organelle that degrades AMPARs, may also respond to synaptic cues to coordinate degradation of membrane proteins such as AMAPRs. We approached the first part of this study by evaluating the distribution and activation of USP8 and how neuronal activity alters USP8 distribution and activation. Using mainly biochemical techniques such as western blot and immunopreciptation as well as immunostaining, we showed that NMDAR signaling is required to spare AMPARs from ubiquitin-dependent internalization and degradation. To elucidate activity-dependent trafficking of lysosomes, we utilized electron microscopy and confocal live imaging of neurons and 2-photon imaging of organotypic hippocampal slices to visualize lysosomes. We found that lysosomes are positioned locally at dendritic spines and can be recruited do a dendritic spine in an activity-dependent manner.

These findings provide evidence for activity-dependent control of a DUB, USP8, and an organelle, the lysosome, at synapses. We showed the dynamic regulation of USP8 is critical in maintaining synapses and scaling during homeostatic plasticity and that neuronal activity can signal to the lysosome to locally degrade membrane proteins. These studies suggest that localized degradation can play an active role in remodeling synapses to facilitate plastic events and help locally maintain cellular homeostasis.