Creation Of A Fatty Acid 2-hydoxylase-knockout Mouse Model
Hiroko Hama
Biochem And Molecular Biologymedical University Of South Carolina
Grant 5R03NS056075-02 from National Institute Of Neurological Disorders And Stroke, IRG: NDBG
Abstract: In higher vertebrates, nerve conduction is greatly facilitated by myelin, a lipid-rich membrane that wraps around the axons. Myelin is formed by oligodendrocytes in the central nervous system, and by Schwann cells in the peripheral nervous system. A number of devastating neurodegenerative diseases are known to cause pathological demyelination. .Myelin consists of approximately 70% lipids and 30% proteins and is highly enriched with two glycolipids, galactosylceramide (GalC) and sulfatide (sGalC, 3-sulfate ester of GalC). A unique feature of these myelin glycolipids is that approximately one half of their fatty acyl chains are 2-hydroxy fatty acids. There are no other mammalian tissues that contain such high contents of 2-hydroxy fatty acids, suggesting that the 2- hydroxyl group has a unique role in myelin. Although much is known about the role of GalC and sGalC in myelination, specific function of the 2-hydroxyl group is not well-understood. The long-term goal of this project is to determine the role of the 2-hydroxy fatty acids of GalC and sGalC in myelination and myelin function. Fatty acid 2-hydroxylation is catalyzed by fatty acid 2-hydroxylase, encoded by the Fa2h gene. It is hypothesized that the Fa2h gene product is responsible for the formation of precursors for 2-hydroxy GalC/sGalC biosynthesis, and that incorporation of 2-hydroxy fatty acids into GalC/sGalC is critical for myelination and myelin maintenance. Current evidence show that Fa2h expression, activity, and the lipid products (2-hydroxy fatty acids) increase during the peak myelination period in postnatal mouse brain. In order to test the hypothesis in vivo, a mouse model that lack Fa2h gene will be developed. The mouse Fa2h gene has been cloned, and a targeting vector has been constructed. The targeting vector is designed to delete exon 1 of the gene, which encodes the N-terminal domain required for the fatty acid 2-hydroxylase activity of Fa2h. Fa2h mRNA, 2-hydroxy fatty acids, and fatty acid 2-hydroxylase activities in the brain of Fa2h-knockout mouse will be determined to confirm successful gene targeting. Another targeting vector for conditional knockout will be constructed, which would be used in case of unsuccessful targeting in ES cells, germline-incompetence of targeted ES cell lines, or embryonic lethality of Fa2h-null mutants. The Fa2h-knockout mouse will be used in future studies to determine the role of 2- hydroxy fatty acids of GalC/sGalC in myelination and myelin function
Project start date: 2007-08-01
Project end date: 2009-07-31
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Grants awarded to Hiroko Hama
FUNCTION AND REGULATION OF MAMMALIAN FAAH
Hiroko Hama
Medical University Of South Carolina 171 Ashley Ave Charleston, Sc 29425
Grant 1P20RR017677-010003 from National Center For Research Resources, IRG: ZRR1
Abstract: The field of sphingolipid biology has exploded because sphingolipids have been established as important signaling molecules [1-3], as well as critical components of membranes [4-7]. A number of hereditary disorders involving sphingolipid metabolism have also been discovered [8, 9]. Specific sphingolipid species involved in distinct biological processes and disorders have been characterized in association with the hydrophilic head groups. In contrast, the roles of structural diversities in the ceramide portion of sphingolipids remain largely uncharacterized, despite the fact that some modifications are known to have dramatic influences on the bioactivities of sphingolipids. The overall goal of this project is to understand the roles of alpha-hydroxy sphingolipids in myelin biogenesis and function. alpha-Hydroxylation of N-acyl chain is a major modification that occurs during de novo sphingolipid synthesis and is catalyzed by an enzyme called fatty acid alpha-hydroxylase (Fig. 1) [10, 11]. alpha-Hydroxy sphingolipids are formed in certain tissues in mammals, most notably in the nervous tissue [12-17]. In central and peripheral nervous systems, characteristic glycosphingolipids are found in myelin, a multi-layered structure formed by the plasma membrane of oligodendrocytes and Schwann cells. These glycosphingolipids are galactosylceramide and its derivative sulfatide, which are highly alpha-hydroxylated (>50%). Mice that do not produce these glycosphingolipids have dysfunctional myelin and show an abnormal behavioral phenotype and a short lifespan [18-20]. The high degree of alpha-hydroxylation in the essential myelin glycosphingolipids suggests that alpha-hydroxylation plays critical roles for myelin biogenesis and function. This hypothesis is testable by modulating the activity of fatty acid (alpha-hydroxylase, which produces the precursor of alpha-hydroxy sphingolipids (Fig. 1). One approach is to identify the fatty acid alpha-hydroxylase gene and manipulate its expression in mice. To this end, the PI identified potential human and mouse fatty acid alpha-hydroxylase genes, named FAAH. We hypothesize that the FAAH gene encodes the fatty acid alpha-hydroxylase that produces alpha-hydroxy glycosphingolipids in brain, and that lack of this enzyme results in defects in myelin biogenesis and function.
Keywords: fatty acid, glycosphingolipid, myelin, oxygenase, protein biosynthesis, protein structure function, enzyme activity, gene expression, genetic regulation, high performance liquid chromatography, immunocytochemistry, in situ hybridization, laboratory mouse, mass spectrometry, northern blotting, nuclear magnetic resonance spectroscopy, polymerase chain reaction, transgenic animal, western blotting
Project start date: 2002-09-26
Project end date: 2007-08-31
Creation Of A Fatty Acid 2-hydoxylase-knockout Mouse Model
Hiroko Hama
Biochem And Molecular Biologymedical University Of South Carolina
Grant 5R03NS056075-02 from National Institute Of Neurological Disorders And Stroke, IRG: NDBG
Abstract: In higher vertebrates, nerve conduction is greatly facilitated by myelin, a lipid-rich membrane that wraps around the axons. Myelin is formed by oligodendrocytes in the central nervous system, and by Schwann cells in the peripheral nervous system. A number of devastating neurodegenerative diseases are known to cause pathological demyelination. .Myelin consists of approximately 70% lipids and 30% proteins and is highly enriched with two glycolipids, galactosylceramide (GalC) and sulfatide (sGalC, 3-sulfate ester of GalC). A unique feature of these myelin glycolipids is that approximately one half of their fatty acyl chains are 2-hydroxy fatty acids. There are no other mammalian tissues that contain such high contents of 2-hydroxy fatty acids, suggesting that the 2- hydroxyl group has a unique role in myelin. Although much is known about the role of GalC and sGalC in myelination, specific function of the 2-hydroxyl group is not well-understood. The long-term goal of this project is to determine the role of the 2-hydroxy fatty acids of GalC and sGalC in myelination and myelin function. Fatty acid 2-hydroxylation is catalyzed by fatty acid 2-hydroxylase, encoded by the Fa2h gene. It is hypothesized that the Fa2h gene product is responsible for the formation of precursors for 2-hydroxy GalC/sGalC biosynthesis, and that incorporation of 2-hydroxy fatty acids into GalC/sGalC is critical for myelination and myelin maintenance. Current evidence show that Fa2h expression, activity, and the lipid products (2-hydroxy fatty acids) increase during the peak myelination period in postnatal mouse brain. In order to test the hypothesis in vivo, a mouse model that lack Fa2h gene will be developed. The mouse Fa2h gene has been cloned, and a targeting vector has been constructed. The targeting vector is designed to delete exon 1 of the gene, which encodes the N-terminal domain required for the fatty acid 2-hydroxylase activity of Fa2h. Fa2h mRNA, 2-hydroxy fatty acids, and fatty acid 2-hydroxylase activities in the brain of Fa2h-knockout mouse will be determined to confirm successful gene targeting. Another targeting vector for conditional knockout will be constructed, which would be used in case of unsuccessful targeting in ES cells, germline-incompetence of targeted ES cell lines, or embryonic lethality of Fa2h-null mutants. The Fa2h-knockout mouse will be used in future studies to determine the role of 2- hydroxy fatty acids of GalC/sGalC in myelination and myelin function
Project start date: 2007-08-01
Project end date: 2009-07-31
1R03NS056075-01 (2007): $73000
Lipid Hydroxylation In Glial Cell Signaling And Myelination
Hiroko Hama
Biochem And Molecular Biologymedical University Of South Carolina
171 Ashley Ave
charleston, Sc 29425
Grant 1R01NS060807-01A1 from National Institute Of Neurological Disorders And Stroke, IRG: CMBG
Abstract: In higher vertebrates, nerve conduction is greatly facilitated by myelin, a lipid-rich membrane that wraps around the axon. A number of devastating demyelinating diseases threat human health, and few effective treatments exist. To develop better treatment for these diseases, we must understand the mechanisms involved in myelination. Myelin is a specialized structure with distinct lipid and protein constituents. Galactosylceramide (GalCer) and sulfatide make up approximately 30% of total myelin lipids, and more than half of these galactolipids contain fatty acid with a hydroxyl group at the C2 position (2-OH galactolipids). No other mammalian tissues contain such high concentrations of 2-OH fatty acids, suggesting that 2-OH galactolipids may play a crucial role in creating the special characteristics of myelin. Despite the extraordinary abundance of 2-OH galactolipids in myelin, there is surprisingly little understanding of the basic biochemistry and physiological role of 2-OH galactolipids. The overall goal of this study is to elucidate the pathway for myelin 2-OH lipids and their roles in myelination, myelin function, and cell signaling. A recently identified fatty acid 2-hydroxylase, FA2H, provides the precursor for the synthesis of myelin 2- OH galactolipids in oligodendrocytes and Schwann cells. FA2H and other enzymes are responsible for the increase in 2-OH very-long-chain (>C20) fatty acid contents in galactolipids during myelination. The first aim of this project is to establish the biosynthetic pathway involved in the unique lipid compositions of myelin galactolipids. Extensive biochemical analyses of FA2H will be performed to determine its physiological substrate, cofactors, and potential feedback mechanisms. Isoforms of fatty acid elongases and dihydroceramide synthases will be identified by a molecular genetic approach. More recently, it was found that reduced FA2H expression via RNAi significantly enhanced motility of D6P2T cells. Cellular 2-OH also partially blocked the upregulation of cyclin-dependent kinase inhibitors, p21 and p27, in response to a stimulus for differentiation. These observations indicate that 2-OH lipids are not only major structural components of myelin, but also function as signaling molecules to modulate cell differentiation and motility. In the second aim, the mechanism of action of 2-OH lipids in cell differentiation and motility will be determined. Transcriptional regulation for p21 and p27 will be investigated to determine the target protein modulated by 2-OH lipids, and the molecular identity of 2-OH lipid species with signaling function will be determined. The third aim is to determine the role of 2-OH galactolipids in myelin function and remyelination in adult brain. The cuprizone- induced demyelination/remyelination will be used to show FA2H is involved in remyelination. Subsequently, newly available conditional FA2H-knockout mice will be used to inactivate FA2H in adult brain. This model will be used to investigate myelin morphology, function, and remyelination in the absence of 2-OH lipids. PUBLIC HEALTH RELEVANCE To develop better treatment for devastating demyelinating diseases, we must understand the mechanisms involved in myelination. This project seeks to unravel the complex pathways for the synthesis of myelin lipids and their roles in myelin maintenance and function, as well as in cell signaling that controls proper myelination. Results obtained from this study will aid in developing better therapeutic agents for neurodegenerative diseases, such as multiple sclerosis
Project start date: 2008-04-01
Project end date: 2013-03-31
REGULATION OF MAMMALIAN FATTY ACID ALPHA-HYDROXYLASE GENE
Hiroko Hama
Medical University Of South Carolina 171 Ashley Ave Charleston, Sc 29425
Grant 5P20RR017677-030003 from National Center For Research Resources, IRG: ZRR1
Keywords: enzyme activity, enzyme induction /repression, fatty acid, oxygenase, gene expression, genetic regulation, clinical research
Project start date: 2004-07-01
Project end date: 2005-06-30
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