ABSTRACT

During early nervous system development, neural stem cells exit the cell cycle and commit to the neuronal phenotype, after which begins the process of neurite outgrowth that gives rise to axons and dendrites. Axons are responsible for establishing and maintaining synaptic connections with target tissues during nervous system development, and are thus essential for the continued development and functionality of the central nervous system (CNS). Folate receptor 1 (Folr1), one of several folate uptake systems, is necessary for the formation of the neural tube, a process that occurs before neurons differentiate and adopt their neuronal morphology. Deficiency of Folr1 in developing vertebrate embryos leads to neural tube defects because of the failure of the neural tube to form and close. Whether Folr1 is important for later stages of spinal cord development is not yet known. Thus, the focus of my thesis is to investigate the role of Folr1 in axon development in the embryonic spinal cord. Here I show that Folr1 is expressed throughout the axons of embryonic spinal cord explants isolated from neural tube stage X. laevis embryos. Using a loss-of-function approach I show that knockdown of Folr1 in embryonic spinal cord explants results in axon retraction, as well as reduced βIII-tubulin enrichment in axons and altered filopodia production and actin dynamics in axonal growth cones. Moreover, I find that the elongation factor eEF1A2, a GTP-binding protein that non-canonically regulates F-actin dynamics, interacts and colocalizes with Folr1 in embryonic spinal cord neuron axons. Knockdown of Folr1 results in depletion of eEF1A2 and phospho(Ser358)-eEF1A2 in axons, and altered localization of eEF1A2 in growth cones. Taken together, these results indicate that Folr1 is important for axon outgrowth during spinal cord development and suggest that interaction of Folr1 with eEF1A2 facilitates proper function of cytoskeletal proteins important for growth cone dynamics.

Revealing the regulatory mechanisms that coordinate neural plate folding and neural tube closure could improve preventative measures for neural tube defects (NTDs). Among the most prevalent and serious birth defects, NTDs occur in 1 in 1000 births in the United States. Multiple causes, both genetic and environmental, have been recognized, including the use of antiepileptic drugs (AEDs) during pregnancy. There is a 10- to 20-fold increase in NTD incidence associated with the use of AEDs during pregnancy, but the teratogenic mechanisms are unknown. The prevalent paradigm that neural activity is not evident at early stages of development suggests that AED teratogenic effects are due to off-target mechanisms. However, recent evidence of embryonic excitability during neural tube formation has challenged this theory and supports the essential need to investigate the presence and regulation of neural activity during nervous system development. Voltage-gated sodium channels (Nav) are one of the most common and efficacious cellular target of AEDs in the mature nervous system yet remain relatively unexplored during neural tube formation. My findings demonstrate that Nav is expressed by Xenopus laevis neural plate cells throughout neural plate folding and neural tube closure and its insertion in neural plate cell membranes increases with the progression of neural tube formation, suggesting that Nav may play an important role in this process. Indeed, I demonstrate that both Nav blockers and Nav-AEDs elicit neural tube defects when exposed to neurulating Xenopus laevis embryos. In addition, Nav blockers and Nav-AEDs comparably reduce neural plate cell calcium activity and reverse the developmental progression of neural plate cell calcium dynamics during neurulation suggesting that AEDs may inhibit calcium transients by impairing Nav function. This work establishes FluoVoltTM membrane potential dye as a useful probe for studying neural plate cell resting membrane potential in Xenopus laevis embryos and demonstrates that the resting membrane potential of neural plate cells is dependent on K+, Na+, and Cl- gradients. Moreover, I find that Nav blockers and Nav-AEDs hyperpolarize neural plate cells. Here I propose a model of mechanisms of Nav-AED induced NTDs. Resting membrane potential of neural plate cells allows for spontaneous activation of Nav which in turn leads to further depolarization of neural plate cells and activation of NMDAR and calcium dynamics. This calcium activity is important for regulating neural plate cell cycle and neural tube formation. Nav-AEDs interfere with Na+ currents and calcium dynamics leading to NTDs. This work significantly contributes to the mechanistic understanding of neural activity regulating neural tube formation to advance the discovery of safer therapeutic options for pregnant women with epilepsy.