DOI
https://doi.org/10.25772/CGV8-2V62
Defense Date
2025
Document Type
Thesis
Degree Name
Master of Science
Department
Biochemistry
First Advisor
Brian Wattenberg
Second Advisor
Sarah Spiegel
Third Advisor
Babette Fuss
Abstract
Canonically known both for structural contributions to lipid bilayers and roles in cell signaling, the sphingolipids comprise a dynamic, multifaceted class of molecules which are studied to understand homeostatic cell biology as well as pathophysiology. Sphingolipids contribute to tissue-specific structures such as myelin, a multilayer membrane that allows for rapid conduction of action potentials along neuronal axons. All sphingolipids are downstream products of the rate-limiting and initiating enzyme in the de novo sphingolipid synthesis pathway, Serine palmitoyltransferase (SPT). Demonstrative of its status as the enzyme that catalyzes a crucial step in a biochemical pathway, SPT activity is strictly regulated. This regulation is accomplished through the ORMDLs, transmembrane polypeptides embedded in the lipid bilayer of the endoplasmic reticulum which function as SPT’s subunits in a native complex. Recently the specific mechanism of the ORMDL regulation of SPT has been established: ceramide, a downstream product of the de novo biosynthetic pathway, binds directly to ORMDL to induce a conformational change that obstructs SPT’s substrate binding site. Consistent with this mechanism, recent studies have revealed that depleting cells of ORMDL dramatically increases sphingolipid levels. In oligodendrocytes it has been specifically shown that deleting one isoform of ORMDL leads to morphological changes (increased thickness) in the sphingolipid-rich myelin membrane and upregulation of myelin associated protein expression. We sought to use our understanding of this mechanism to lay the foundation for clinical interventions that involve raising SPT’s catalytic activity by way of abrogating ORMDL’s inhibitory function. These interventions could be particularly beneficial to patients with skin sphingolipidoses, where application of a small molecule enzyme activator could be beneficial (and topically administered), and in the CNS, where a targeted intervention in the SPT/ORMDL pathway could allow patients with demyelinating diseases to produce more of the sphingolipid-rich myelin membrane. To heighten SPT activity in vitro, we investigated molecules that could interfere with the direct binding of ceramide via competitive binding to ORMDL. Such competitive binding—in the classical biochemical sense of the term—involves the stable occupation of the ORMDL site by a molecule incapable of inducing an inhibitory conformation of SPT/ORMDL, thus blocking inhibition. Our search for a competitor was guided by our newly validated in silico molecular docking protocol which—in addition to strengthening previous mechanistic studies on ORMDL’s regulation of SPT—fascinatingly predicted that non-native stereoisomers of ceramide could bind with similar affinity to ORMDL3 as the native D-erythro ceramide. Using an established assay of SPT activity, we tested the effect of ceramide’s stereoisomers and found that L-erythro ceramide weakens the inhibition of SPT from D-erythro ceramide in an ORMDL-dependent manner. Additionally, we hypothesized that raising SPT activity by deleting one of the ORMDL isoforms in mouse oligodendrocytes would alter the repair of demyelinated axons in a model of Multiple Sclerosis (MS). We utilized a conditional, inducible knockout of ORMDL3 and the Experimental Autoimmune Encephalomyelitis (EAE) model to test this hypothesis. Consistent with our hypothesis, we found that female mice with the knockout had significantly reduced neurological symptoms throughout the course of the experiment compared to wild type littermates—interestingly, we saw no differences in males. Though we have yet to substantiate this phenomenon with data showing knockout-specific protective changes in the myelin membrane or immune response, we believe that this data identifies sphingolipid biosynthesis in oligodendrocytes is an area that warrants further study for the treatment of MS.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
5-6-2025