Date of Award

10-30-2020

Publication Type

Master Thesis

Degree Name

M.Sc.

Department

Biological Sciences

Keywords

activity-dependent synaptic growth, cAMP-PKA pathway, cholesterol, D4H biosensor, neuromuscular junctions, synaptic growth

Supervisor

Jeffrey S. Dason

Rights

info:eu-repo/semantics/embargoedAccess

Abstract

Cholesterol is a versatile molecule that determines the biophysical properties of cellular membranes and regulates various signalling pathways. Cholesterol has also been implicated in synaptic development. In vitro studies have shown that reducing cholesterol levels can increase or decrease neurite outgrowth in different types of cultured neurons. However, the underlying mechanisms of how reduced cholesterol content of membranes affects synaptic growth have not been explored in detail. Cholesterol has been shown to regulate the cAMP-protein kinase A (PKA) pathway in non-neuronal cells. The cAMP-PKA pathway is known to be an important regulator of synaptic development. My primary objective was to elucidate the cellular mechanisms by which cholesterol regulates synaptic growth. I extracted cholesterol from Drosophila larval neuromuscular junctions in controls and mutants known to be involved in cAMP-PKA signalling and then examined the effects that this had on synaptic growth. First, I found that cholesterol is required for the normal synaptic growth. Second, chronic and acute cholesterol extraction reduced PKA activity. Next, I extracted cholesterol from mutants with elevated cAMP levels and found that synaptic growth was also reduced in these mutants, suggesting cholesterol may be acting downstream of cAMP. Furthermore, I found that cholesterol was required for activity-dependent synaptic growth, which is known to be regulated by cAMP-PKA signalling. Lastly, I constructed a vector based on domain four of the theta-toxin produced by Clostridium perfringens to serve as a genetically encoded cholesterol biosensor to determine the distribution of cholesterol at the Drosophila larval neuromuscular junction. Overall, my results suggest that cholesterol-dependent effects on synaptic growth may be due to changes in cAMP-PKA signalling. My findings demonstrate that changes in the cholesterol content of neurons can affect synaptic development, which in turn could result in synaptic dysfunction.

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