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.

Ubiquitin-dependent receptor endocytosis and subsequent endocytic sorting to the lysosome for degradation plays an essential role in synaptic plasticity by changing the number of membrane receptors at the synaptic sites. However, our knowledge of this pathway on the overall synaptic membrane protein population is still rudimentary. Here, we identify surface synaptic membrane proteins as candidate ubiquitinated targets of the lysosome. We show that the lysosomal inhibition increases surface synaptic membrane protein ubiquitination in neurons. We combine the isolation of biotinylated cell surface membrane proteins, lysosomal perturbation and mass spectroscopy analysis to generate the first large-scale proteomic analysis of the surface synaptic proteome as candidate ubiquitinated targets of the lysosome. After 12 hours treatment, we compared biochemically purified synaptic cell membrane protein fractions between normal and lysosome-perturbed conditions through tandem mass spectrometry. A total of 1600 proteins were identified and quantified, including 539 plasma membrane proteins. Out of these plasma membrane proteins, we found that 105 of them were upregulated, and 207 were downregulated, while 226 did not significantly change in protein level. The proteins with altered pattern include proteins involved in regulating cell adhesion, cellular homeostasis, synaptic transmission, and cellular localization. This suggests that the ubiquitin-dependent lysosomal degradation pathway is important in maintaining synaptic function.

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.

Prion diseases are rapidly progressive neurodegenerative diseases characterized by spongiform degeneration, gliosis, synaptic loss, and neuronal dystrophy. Prion disease is caused when the cellular glycosylphosphatidylinositol (GPI)-anchored prion protein, PrPC, misfolds into an aggregated conformer, known as PrPSc. PrPC has an unstructured flexible N-terminus and a structured C-terminus with two N-linked glycans. Several human prion diseases are caused by mutations in the N-terminus of PrPC. For example, some cases of Gerstmann-Straüssler- Scheinker disease or Creutzfeldt-Jakob disease are caused by insertions in the octapeptide repeat region of the N-terminus. Furthermore, the untethered N-terminus of PrPC can act as a neurotoxic effector domain. Mice expressing only the N-terminus of PrPC show altered interactions between the truncated PrPC and endoplasmic reticulum proteins, which results in neurotoxicity. To understand how the N-terminus impacts PrPC function and neurodegenerative disease progression, we generated a knock-in mouse model that modifies the N-terminus of PrPC by substituting glycine 93 to asparagine, allowing the N-terminus to be glycosylated and potentially untethered. This mutation causes spontaneous neurodegeneration without the presence of PrPSc aggregates. In vitro experiments support a role for excitotoxicity in the neurodegenerative phenotype of the Prnp93N mice. Hippocampal and cortical primary neuron cultures from Prnp93N mice display dendritic beading, which is characteristic of excitotoxic stress. The excessive dendritic beading in the Prnp93N neurons was rescued using a glutamate receptor antagonist implicating signaling through glutamate receptors that are driving the excitotoxic insult. In vivo experiments also indicate a role for excitotoxicity in a brain specific region. Hippocampi of p26 Prnp93N mice have higher phosphorylated Ca2+/calmodulin-dependent protein kinase II (CaMKII) at threonine 286 (Thr286). Calcium ion binding to CaMKII activates the kinase and promotes autophosphorylation at Thr286 to constitutively activate CaMKII. Hippocampi of p26 Prnp93N mice also show higher phosphorylated glutamate receptors, indicating increased channel conductance when compared to PrnpWT mice. Cortex samples do not show differences. Together, this data suggests neuronal activation differs between the cortex and the hippocampus in the Prnp93N mice. This study supports the hypothesis that enhanced excitotoxic signaling involving receptor contributes to this neurodegenerative phenotype.

The establishment and stabilization of proper synaptic connections is a vital step in the maturation of cortical circuits. The prevention of these processes can lead to downstream developmental delays and unstable connections in the adult. We show that astrocyte expressed glypican 5 (Gpc5) is necessary for the refinement and strengthening of thalamocortical synapses in the mouse primary visual cortex binocular zone (V1b) during the critical period. Using electron microscopy, we show that in the absence of Gpc5, thalamocortical synapses show distinct structural immaturity, including smaller boutons and weaker synapses during the critical period. This structural immaturity is accompanied by a delay in the developmental incorporation of GluA2-containing calcium impermeable AMPARs at intracortical synapses, as observed using immunohistochemistry. Upon reaching adulthood, mice lacking astrocytic Gpc5 demonstrate intact synaptic composition but exhibit increased levels of ocular dominance plasticity, potentially as a result of destabilized thalamocortical synapses. The work in this thesis suggests that astrocytic Gpc5 is necessary for the stabilization of thalamocortical terminals during development and in the adult.

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.

Mucopolysaccharidosis type IIIC (MPS IIIC) is a severe neurodegenerative lysosomal storage disease stemming from the loss of function of the lysosomal transmembrane protein Heparan glucosamine N-acetyltransferase (HGSNAT). This leads to the accumulation of the Glycosaminoglycan: Heparan Sulfate. Currently there are no available treatments for this disease. To address this, we developed a new mouse model which displays key disease features such as GAG buildup, Splenomegaly, neurological impairment, and the presence of disease specific non reducing end carbohydrates. In order to investigate a new therapeutic approach for this disease, we transplanted hematopoietic stem and progenitor cells (HSPCs) isolated from Wildtype mice into Hgsnat -/- mice and observed differences between the treated and untreated Hgsnat -/- mice. Transplanted HSPCs differentiated into macrophages in peripheral tissues and microglia-like cells in the brain. The exogenous supply of HSPCs resulted in partial restoration of HGSNAT expression and enzymatic activity which led to a notable reduction in MPS IIIC specific non reducing end carbohydrates within the Hgsnat -/- mice treated with WT HSPCs compared to the untreated Hgsnat -/-mice as well as Hgsnat -/-mice treated with HSPCs harvested from fellow Hgsnat -/-mice. Additionally, WT HSPC transplantation was able to improve neurological deficits, decrease splenomegaly and urine retention in the Hgsnat -/-mice. Histological analysis of the diseased mice kidneys revealed the presence of glomerular hyaline bodies with focal fibrosis and sclerosis which were mitigated in the mutant mice treated with WT HSPCs. The findings from this study provide evidence for the potential therapeutic benefits that WT HSPC transplantation offers MPS IIIC and could serve as an early step toward HSPC gene therapy becoming a clinical approach for rare hereditary diseases such as MPS IIIC.

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.