Hiroko Hama - US grants

enzyme activity; oxygenases; enzyme induction /repression; fatty acids; genetic regulation; gene expression; clinical research;

[unreadable] DESCRIPTION (provided by applicant): 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. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): 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.

DESCRIPTION (provided by applicant): The leukodystrophies are a group of disorders that affect myelin. Most of the leukodystrophies are hereditary and cause progressive degeneration of the white matter. Currently, there are no cures for any of the leukodystrophies. The focus of this project is a recently identified leukodystrophy caused by mutations in the fatty acid 2-hydroxylase (FA2H) gene, which result in lack of a type of myelin lipids. Because this disease is caused by a lipid deficiency, there is a distinct possibility that it could be treatable by replacing the missing lipids. The ultimate goal of this study is to develop synthetic lipid therapeutics that can rectify the deficit in patients'myelin lipids, thereby preventing degeneration of the white matter. This project is a preclinical study aimed at developing potential lipid therapeutics using a Fa2h knockout mouse model. FA2H is a lipid biosynthetic enzyme responsible for the synthesis of major myelin sphingolipids. Galactose-containing sphingolipids (galactolipids) make up approximately 30% of total myelin lipids. More than half of the myelin galactolipids contain 2-hydroxy fatty acids (hFA) as their N-acyl chains (hFA-galactolipids). FA2H is the enzyme responsible for the synthesis of the precursor hFA in myelin-forming cells. FA2H deficiency results in loss of hFA in myelin galactolipids, which eventually leads to degeneration of the white matter. There is a possibility that the disabling leukodystrophy could be prevented if myelin-forming cells are exogenously provided with sufficient hFA. To explore this possibility, synthetic hFA-lipids will be administered to Fa2h- knockout mice through the diet. The experiments are designed to address the following three questions: 1) Are the hFA-lipids absorbed? 2) Are the exogenous hFA incorporated into myelin galactolipids? 3) Does long-term administration of the hFA-lipids cause any adverse effects? The study will be conducted in three phases. In the initial phase, Fa2h+/+ and Fa2h-/- weanlings will be fed milled chow supplemented with various synthetic triacylglycerols containing hFA (hFA-TAG) for 5 days to determine whether the test lipid can be absorbed. Blood samples will be collected to determine plasma hFA levels. Those hFA-TAG that are absorbed will be tested for delivery of hFA to myelin. In this phase of the study, Fa2h+/+ and Fa2h-/- weanlings will be fed milled chow supplemented with the hFA-TAG for 4 weeks. At the end of the 4-week period, brain lipids will be analyzed to determine the presence of hFA-galactolipids. Positive compounds will then be tested for long-term effects. If successful, this study will lead to development of potential therapeutics that will prevent and cure the leukodystrophy caused by FA2H deficiency. PUBLIC HEALTH RELEVANCE: Children with FA2H deficiency suffer from a devastating leukodystrophy. Fortunately, the cause of disease has recently been identified: the patients lack a type of fatty acids that make up myelin sheath. The goal of this project is to develop therapeutics to provide the missing fatty acids to patients'myelin to prevent and cure the disease.

ABSTRACT The goal of this project is to study the role and mechanisms by which host sphingolipids are involved in controlling the infection caused by the pathogenic fungus Cryptococcus neoformans (Cn). A rapidly emerging area of research is the study of the role of sphingolipids in the regulation of infectious diseases (Reviewed in18, 22). Most of these studies have focused on the role of microbial sphingolipids in the ability of the microbe to cause infection. Very few studies addressed if and how host sphingolipids are also involved in the regulation of microbial pathogenesis, and most of these studies have focused on bacterial or parasitic infections (reviewed in18, 22). Although some sphingolipids have been linked to antibacterial activity of phagocytic cells16, 42, 43, 59, nothing is known about the role of host sphingolipids against fungal infections. Since phagocytic cells, such as macrophages and neutrophils, are the first line of defense against Cn infection, the regulation of their cellular processes may affect their response to the fungus, and thus, determine whether they can or cannot control the development of cryptococcosis. One of the host sphingolipid-metabolizing enzymes shown to regulate immune responses is sphingomyelin synthase (SMS) encoded by two genes, SMS1 and SMS217, 30, 36, 39. SMS transfers a choline phosphate moiety from phosphatidylcholine (PC) to ceramide, therefore producing sphingomyelin (SM) and diacylglycerol (DAG)28, 62, 65. Very interestingly, the lipids regulated by SMS have been implicated in the activation of pro-inflammatory responses, suggesting that the regulation of SMS activity in immune cells may assume a critical role in controlling infections. In our preliminary studies we found that: 1) inhibition of SMS activity profoundly impairs the ability of neutrophils to kill Cn cells by affecting extracellular killing in absence of phagocytosis;2) SMS regulates production of DAG at the Golgi;3) DAG at the Golgi regulates protein secretion through a protein kinase D (PKD)-mediated mechanism;4) inhibition of PKD blocks extracellular killing of Cn;and 5) inhibition of either SMS or PKD activity significantly decreases secretion of antimicrobial peptite(s) such as α-defensin. Based on these observations, we hypothesize that SMS activity plays a key role in controlling Cn infection through a DAG-PKD-antimicrobial peptide(s) secretion pathway. Thus, we propose the following aims: 1) To determine the role of SMS activity during Cn infection;and 2) To define the mechanism by which SMS activity regulates the extracellular killing of Cn. By studying how host sphingolipids regulate the extracellular killing activity of neutrophils against Cn, we will provide new insights not only for a better understanding of fungal pathogenesis but also for the development of new therapeutic strategies against this and potentially other fungal microbes. The studies that we propose in this application will reveal new regulatory mechanisms involved in the killing of Cn by neutrophils. We will identify the nexus between SMS, PKD and infection.