Phosphatase PTP-3 regulates AMPA receptor transport in Caenorhabditis elegans, The

2021 Summer.Includes bibliographical references.Glutamate mediates the majority of excitatory neurotransmission by activating the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype of ionotropic glutamate receptors. AMPAR trafficking, which includes local synaptic trafficking and long-distance transport, can be one of many cellular pathways important for cognition. Since the majority of AMPARs are made at the cell body, this exerts a challenge on neurons to correctly transport receptors to often far-reaching synapses. To ensure correct synapse function, the interplay between long-distance AMPAR transport, delivery, removal, and retention need to be tightly regulated. However, how long-distance transport mechanisms communicate with local synaptic delivery and removal is unknown. Previous work has shown a critical role for the receptor-type protein tyrosine phosphatase (RPTP) leukocyte common antigen-related protein (LAR), in regulating AMPAR numbers at synapses. However, these studies have not identified a mechanism for this process. Therefore, my thesis sets out to test how AMPAR transport impacts local synaptic trafficking and the role that the C. elegans homologue of vertebrate LAR, Protein Tyrosine Phosphatase 3A (PTP-3A), has on this regulatory pathway. Chapter 1 is an introduction and its purpose is to provide background knowledge and scientific context for the main questions of my thesis. In Chapter 2, we investigate the question of how does glutamate receptor transport impact local synaptic glutamate receptor numbers. Tyrosine phosphorylation is known to play an important role in glutamate receptor trafficking at the synapse, but not much is known about tyrosine phosphorylation on glutamate receptor transport. The vertebrate phosphatase LAR is known to be important in glutamate receptor transmission in cell culture, but its role in glutamate receptor transport is unknown. Here we investigate the role of the sole C. elegans LAR-RPTP homologue, PTP-3A, on glutamate receptor transport and how this affects local synaptic delivery. We show that PTP-3A mutants display decreased transport as well as decreased delivery of Glutamate Receptor-1 (GLR-1) to synapses. Since PTP-3A is a large structure, 3 IgG domains, 9 fibronectin domains, and 2 phosphatase domains, we sought out to determine what domains were necessary for GLR-1 transport. Domain analysis of LAR revealed that the phosphatase domain is not required for GLR-1 transport but is required to stabilize GLR-1 at synapses. Surprisingly, the Ig-like external domains are sufficient to cell-specifically rescue GLR-1 transport. Interestingly, LAR mutants exhibit decreased short-term and long-term memory whereas mutants lacking the phosphatase domain only show decreased long-term memory. This could be due to a mechanism where efficient GLR-1 transport is sufficient to sustain the synaptic receptor pool during short-term synaptic activity, but stabilization of GLR-1 at synapses is required long-term consolidation. Taken together, our results show a critical role of LAR in long-distance synaptic AMPAR transport. Chapter 3 seeks to identify a role of a known regulator of vertebrate LAR, liprin-α, in glutamate receptor transport. Vertebrate liprin-α is known to bind LAR and correctly localize it to synapses. Previously, the C. elegans sole liprin-α homologue, SYD-2, has only been identified as a regulator of active zone maintenance presynaptically. However, vertebrate liprin-α is known to colocalize with postsynaptic proteins such as PSD-95, GRIP-1, LAR, and GluA2. Since LAR and liprin-α colocalize at the synapse, we reasoned that this interaction might happen in C. elegans as well and that SYD-2 might be in the same regulatory pathway as PTP-3A. Therefore, we asked if loss of SYD-2 would cause a defect in GLR-1 localization in vivo in C. elegans. We show that loss of SYD-2 leads to ~1.5-fold increase in GLR-1 transport but has a ~70% reduction in synaptic and surface GLR-1. This leads to an interesting model where synaptic activity might not be directly correlated with GLR-1 transport. Also, our data suggest that SYD-2 might have additional synaptic roles other than correctly localizing PTP-3A. Finally, Chapter 4 aims to discuss the impact of the data I have generated in my thesis and how experiments from each chapter complement each other and how these have opened new experimental paths. Overall, the work in my dissertation expands on the limited knowledge of how AMPAR transport and synaptic trafficking interact to control synaptic AMPAR numbers. It furthers our knowledge of the role of the phosphatase PTP-3A/LAR in regulating excitatory synaptic maintenance. It also challenges the idea which recent studies have shown that correlates decreased synaptic activity with decreased AMPAR transport. Altogether, it outlines the general importance of understanding how AMPAR transport relates to synaptic plasticity and behavior