Author ORCID Identifier

https://orcid.org/my-orcid?orcid=0000-0002-7418-096X

Defense Date

2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Biochemistry

First Advisor

Dr. Binks Wattenberg

Abstract

The ORMDLs are regulatory subunits of the serine palmitoyl transferase (SPT) enzyme. Despite the significant amount of research that has been done to understand how ORMDLs regulate SPT activity, there are still gaps in the field that need to be explored. Specifically, two gaps exist: first, why cells require three ORMDL isoforms to regulate SPT, and second, how ORMDLs are extracted from the SPT complex for their degradation and how they are delivered to autophagy to maintain their regulatory turnover. The second chapter of this thesis is dedicated to understanding the first missing gap. The hypothesis is that each ORMDL isoform can regulate SPT activity by sensing different ceramide levels. To test this hypothesis, we generated cell lines using HeLa cells in which two of the three ORMDLs were deleted using CRISPR/Cas9 gene editing. The results showed that each ORMDL isoform can indeed regulate SPT activity by sensing different ceramide levels. The third chapter of the thesis aims to uncover the molecular mechanism by which the ORMDLs bound to SPT undergo regulatory turnover in response to low cellular sphingolipid levels. For the first time, we discovered that low cellular ceramide levels accelerate the constitutive turnover of the SPT-bound ORMDLs. The half-life of SPT-bound ORMDLs is approximately 12 hours, but in low cellular ceramide conditions, the half-life is reduced by approximately 4 hours. The results suggest that both constitutive and regulatory turnover of SPT-bound ORMDLs is maintained by autophagy, but the exact mechanism has yet to be discovered. The final chapter of this thesis focuses on understanding the functional importance of the ORMDL3 isoform in CNS myelination. The hypothesis is that the regulation of sphingolipid biosynthesis by the ORMDLs in oligodendrocytes is crucial for proper myelination. To test this hypothesis, we generated oligodendrocyte-specific ORMDL3 conditional KO mice using the Cre/Lox system. The results showed a coordinated increase in larger caliber axons and increased myelin thickness in the optic nerves of 30-day-old ORMDL3 cKO mice, as demonstrated by electron microscopy. Overall, the studies suggest that sphingolipid biosynthesis is a key control mechanism that coordinates both lipid and protein synthesis in oligodendrocytes during myelination.

Rights

© USHA SARASWAT MAHAWAR

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

5-6-2024

Available for download on Saturday, May 05, 2029

Included in

Neurosciences Commons

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