Date of Award

2007

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

College

College of Graduate Studies

First Advisor

L. M. Obeid

Second Advisor

Michael J. Kern

Third Advisor

Maurizio Del Poeta

Fourth Advisor

Chiara Luberto

Fifth Advisor

Hiroko Hama

Abstract

Myelin is formed by oligodendrocytes in the central nervous system and Schwann cells in the peripheral nervous system. This unique membrane is comprised of approximately 70% lipid and 30% protein. The high lipid content is thought to be vital for its insulatory function. Galactosylceramide (GaICer) and sulfatide make up approximately 30% of total myelin lipids, with more than half of these galactolipids containing fatty acids hydroxylated at the C[subscript 2] position (2-hydroxy fatty acids). Despite their high abundance, very little is know about the biosynthesis of these 2-hydroxy galactolipids, and specific functions of the 2-hydroxyl group in myelin galactolipids remain speculative. To fill this gap, we recently cloned and characterized a human fatty acid 2-hydroxylase gene, FA2H, that is highly expressed in brain. To study the roles of fatty acid 2-hydoxylase and 2-hydroxy sphingolipids in the brain and other tissues, we have developed highly sensitive methodologies to measure in vitro fatty acid 2- hydroxylase and free 2-hydroxy fatty acids by gas chromatography/mass spectrometry (GC/MS). Utilizing these novel methods, here we demonstrate that FA2H is required for the formation of 2-hydroxy fatty acids (precursors of 2-hydroxy galactolipids) in the central and peripheral nervous systems, and 2-hydroxylation of free fatty acids is the first step for the biosynthesis of 2-hydroxy galactolipids. During the course of the study we developed FA2H knockdown using siRNA and shRNA. RNAi against FA2H revealed unexpected cellular phenotypes indicative of altered differentiation. Cells were 2.8-fold more migratory, exhibited increased proliferation, and were strongly resistant to cAMP induced differentiation. These findings open an exciting area of research involving 2- hydroxy lipids in cell growth, migration, and differentiation.

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