Author ORCID Identifier
https://orcid.org/0000-0002-3390-401X
Date Available
4-12-2025
Year of Publication
2024
Degree Name
Doctor of Philosophy (PhD)
Document Type
Doctoral Dissertation
College
Medicine
Department/School/Program
Neuroscience
First Advisor
Dr. Salvatore J. Cherra
Abstract
Synaptic dysfunction is a major contributor toward the development of many neurological disorders. For proper development, synapses require a series of signaling pathways to guide them, but not all the mechanisms underlying these processes are understood. Here, we used the neuromuscular junction of Caenorhabditis elegans as a model circuitry for examining underlying pathways that mediate proper development of synapses. We hypothesized that the C. elegans RapGEF2 and RapGEF6 orthologue, PXF-1, contributed toward development of cholinergic synapses at the neuromuscular junction. Using behavioral assays and fluorescence microscopy, we found that pxf-1 mutant animals had decreased neuromuscular junction function and decreased synaptic vesicle intensity with no changes to the active zone markers. We observed no changes to intensity or density of synaptic vesicles in GABAergic synapses. Based on rescue through pan-neuronal expression of the actin cytoskeleton modulator, WVE-1, we determined that the probable mechanism for PXF-1 is through actin formation or reorganization. We then investigated the G-proteins involved in this pathway. We utilized loss-of-functions mutations in rap-1 and rap-2, the canonical GTPases for PXF-1, and whether they were involved in this pathway. We found that RAP-1 but not RAP-2 may be necessary for the synaptic function of PXF-1. Additionally, a constitutively active form of RAP-1 was able to rescue pxf-1 deficits. Moving to known G-proteins and modulators that are known to influence actin-mediated synaptic organization, we identified RAC-2 as a downstream target of PXF-1 with RAC-2 activity decreased in pxf-1 mutants in vivo. To examine whether this interaction from RAP-1 to RAC-2 is direct, we investigated whether the TIAM-1, a RacGEF whose subcellular location is known to be influenced by RAP-1 activation, was also involved in this pathway. By using constitutively active forms of RAC-2 and RAP-1 and TIAM-1 loss of function mutations, we were able to determine PXF-1 may activate RAC-2 via RAP-1 to TIAM-1 signaling. Inversely, we sought out a GTPase activating protein that may be able to ameliorate the deficits observed. We found that inhibition of GAP-1, a RASA3 orthologue, was able to rescue the phenotypes in pxf-1 animals. These findings indicate the participation of Ras superfamily signaling in synaptic development either in tandem with pxf-1 or parallel. Together, the results presented in this dissertation highlight the necessity of balanced G-protein signaling in the nervous system.
Digital Object Identifier (DOI)
https://doi.org/10.13023/etd.2024.226
Funding Information
This research was support in part by grants from the National Institute of Health: R00 (NS097638) in 2019, Ruth L. Kirschstein Predoctoral Fellowship (NS129159-01A1) in 2023, and R01(NS129668-01A1) in 2023.
Recommended Citation
Lamb, Reagan EM, "Caenorhabditis elegans RapGEF promotes synaptic development through a small G protein signaling cascade" (2024). Theses and Dissertations--Neuroscience. 34.
https://uknowledge.uky.edu/neurobio_etds/34
