DOI
https://doi.org/10.25772/K9TS-FG87
Defense Date
2023
Document Type
Thesis
Degree Name
Master of Science
Department
Biology
First Advisor
Dr. Jason Newton
Second Advisor
Dr. Santiago Lima
Third Advisor
Dr. Derek Prosser
Fourth Advisor
Dr. Sarah Rothschild
Abstract
Niemann Pick Type C (NPC) is a recessively inherited lysosomal storage disorder for which there is currently no major treatment. It is widely acknowledged that the pathology of NPC is caused by a trafficking defect which leads to an accumulation of cholesterol in the late endosome/lysosome compartment (LE/L). In addition to cholesterol, multiple species of sphingolipids are observed to accumulate as well in the LE/L. Specifically, an accumulation of sphingosine is observed in NPC disease and is likely attributed to the decreased activity of an enzyme, sphingosine kinase 1 (SphK1), which is responsible for phosphorylating sphingosine into an important pro-survival signaling molecule called sphingosine-1-phosphate (S1P). Previous work has shown that either the overexpression of SphK1 or the addition of nM concentrations of S1P can correct deficiencies observed in NPC models, however the impact of decreased S1P and the signaling cascades they regulate via the activation of 1 of 5 transmembrane S1P receptors are incompletely described. Accordingly, we sought to determine the baseline levels of the S1P receptors (S1PRs) in NPC1 mutant and NPC1 knockout (KO) cells, as well as the biological consequences of the dysregulated expression of the S1PR’s in NPC disease. In this study, a significant increase in S1PR1, and S1PR2 mRNA was observed via reverse transcriptase (RT) qPCR in NPC1 KO cells, as well as in mouse brain and liver tissue. Conversely, western blot analysis of NPC1 KO HeLa cells shows no difference in protein expression of S1PR1 or S1PR2. Following gene and protein expression analysis, the cellular localization of S1PR1 and S1PR2 were analyzed via fluorescence microscopy. Initial results showed that NPC1 KO cells have no change in transmembrane S1PR1, but a decreased amount of transmembrane S1PR2. The functionality of S1PR1 and S1PR2 was assessed by treating NPC1 KO cells with agonist and antagonist drugs for S1PR1 and S1PR2 and utilizing western blots to qualitatively and quantitatively analyze the presence and activation of the phosphoinositide-3-kinase (AKT) and mitogen-activated protein kinase (ERK) signaling cascades. Initial results indicated that NPC1 KO cells treated with CYM-5520 (S1PR2 agonist) demonstrated a significant decrease in activation of the AKT pathway, and that treatment with JTE-013 (S1PR2 antagonist) restored activation of the AKT pathway. These results indicate that S1PR2 blockage may provide potential therapeutic benefits, however further studies are needed to elucidate specific molecular interactions involved in the regulation of the AKT pathway by S1PR2.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
5-10-2023