Steven A. Farber - US grants

DESCRIPTION Phospholipase A2 (PLA/2) activity results in the synthesis of a number of eicosanoids, potent lipid mediators that play a role in inflammation, mitogenesis and metastasis of cancer cells, and are likely to be important during development. The substrate for the synthesis of all eicosanoids is arachidonic acid (AA), a molecule generated by kinase C (PKC) and annexins (ANX). ANX I is phosphorylated by growth factor receptors, and is a PLA/2 inhibitor. While there has been extensive study of the action of PLA/2 and its modifiers in cell culture, very little is known about their roles in the more complex context of a developing embryo. To investigate PLA/2 function in vivo, I chose a well characterize vertebrate system, the zebrafish Danio rerio, because its embryos are abundant and optically clear. This facilitates both the visualization of gene expression through whole mount in situ hybridization and the development of fluorescently-quenched lipid probes that can directly report PLA/2 activity than non-mitotic cells. I will examine PLA/2's role in cell signaling during development in both wild-type and mutant embryos identified by a biochemical screen for altered PLA/2 activity performed during the first two years of funding. I will examine the effect of over- expression of zebrafish annexins on embryonic PLA/2 activity and the phenotype consequences. I will examine the effect of over expression of zebrafish annexins on embryonic PLA/2 activity and the phenotypic consequences. My long-term objective is to understand the role of PLA/2 and its modifiers during development and thereby provide insight into the pathology associated with their aberrant activation.

DESCRIPTION: (Applicant's Abstract) Although morphology-based genetic screens have identified mutations that affect specification and patterning of zebrafish embryonic structures, few reagents have been applied to assess physiological functions in vivo. We produced fluorescent lipids that are substrates for phospholipase A2 (PLA2), an important signaling and digestive enzyme present in the cytoplasm and brush border of the intestinal epithelium. Cleavage of these lipids by PLA2 increases or shifts their wavelength of fluorescent emission, thereby revealing localized enzymatic activity in live animals. When administered to optically transparent zebrafish larvae, these lipids provide a real-time visual assay of PLA2 activity. Fluorescent lipids ingested by day 5 post-fertilization zebrafish larvae produce intense gallbladder fluorescence, a finding we have shown reflects lipid processing by intestinal PLA2 and subsequent transport through the hepatobiliary system. Since the rate and degree of gallbladder fluorescence are easily observed in zebrafish larvae, these reagents provide a sensitive readout of digestive physiology that in the context of a mutagenesis screen can identify genes that influence lipid processing. We administered fluorescent lipids to mutagenized 5-day post-fertilization zebrafish larvae and have identified one mutation that perturbs intestinal phospholipase activity. The work described in this proposal outlines our plan for a large scale mutagenesis screen using these reagents. By identifying mutations that perturb lipid metabolism, we hope to recover genes that regulate intestinal and hepatobiliary development as well as PLA2, a gene with an imprint role in inflammation, hemostasis and cell proliferation. Genetic characterization of these developmental and physiological processes has important implications for research related to congenital diseases of the intestine and liver, cancer, and cardiovascular and inflammatory diseases.

A number of widespread diseases that impact both quality of life and longevity are influenced by dietary fat. One major impediment to improving our understanding of lipid metabolism and its related disorders is that so few metabolic studies have been carried out in live organisms. As a result, the dynamic regulatory signals that coordinate the absorption and transport of fatty acids (FA) in vivo, such as their uptake from the intestinal lumen and secretion to the rest of the body, are not fully understood. To this end, we have developed an animal model of dietary fat (triacylglycerol) metabolism that can track FAs from their site of absorption at the intestinal brush border of 6-day old zebrafish larvae, through the circulatory system, and ultimately to lipid drops within a variety of organs. While it is well known that intestinal enterocytes are the main absorptive cell that can take in large amounts of dietary lipid and export it in the form of lipoproteins to the rest of the organism, many questions remain regarding the precise cell biological processes involved. The optically clear 6-day old zebrafish larva is ideally suited to address these gaps in our understanding of vertebrate intestinal lipid absorption and processing. We have developed methods to feed larvae a high-fat diet and subsequently characterize intestinal enterocytes by electron microscopy (EM). This [unreadable]supersized[unreadable] diet produced prodigious subcellular lipid accumulations that had all the morphological features of lipid drops. While EM analysis was informative, it precluded real-time live imaging of fed larvae. To address this limitation, we included fluorescent lipids in our feeding protocol that enabled visualization of intestinal lipids in live animals. We developed microscopy techniques to visualize lipid accumulations at the whole organ level (1x objective) and at the subcellular level (63x objective). Varying the type of fluorescent lipid enabled us to elucidate two specific lipid-dependent transport processes (cholesterol vs triacylglycerol). We also found that very short chain fluorescent FAs are processed profoundly differently than medium and long chain FA in that they accumulate exclusively in the ductile networks of the liver and pancreas. The recent availability of zebrafish lines with known mutations provides a unique opportunity to functionally annotate the vertebrate genome. A recent community-wide meeting set out broad guidelines for a strategy to phenotype large numbers of zebrafish lines generated from the existing TILLING consortiums and stressed the importance of novel screening tools. We propose to screen existing mutant lines using our high-fat feeding paradigm with a variety of fluorescent reagents. The work described focuses on assays of digestive organ function that go significantly beyond previous approaches that described mutations from only the perspective of organ development and patterning. It is expected that we will identify genes that influence FA and lipoprotein metabolism, making them potential targets for future therapeutics.

DESCRIPTION (provided by applicant): Aberrant lipid metabolism contributes to the etiology of multiple human diseases including cardiovascular disease (CVD), obesity, and insulin resistance (IR). A major impediment to improving our understanding of lipid metabolism and its related disorders is that so few metabolic studies have been carried out in live organisms. As a result, the dynamic regulatory signals that coordinate absorption and transport of fatty acids (FA), and morphogenesis and fat storage in adipose tissues, remain unclear. To address this gap, the assembled research team has pioneered methods to image lipid uptake, transport and storage, within complex organs composed of many cell types in live zebrafish. The Farber lab has established tools to visualize the cellular dynamics of dietary FA in zebrafish larvae, while the Rawls lab has developed complementary methods for using vital fluorescent lipophilic dyes to visualize zebrafish adipose tissues. These state-of-the-art in vivo optical reporters provide a comprehensive view of organ physiology not revealed in previous studies of just organ development. We propose to use these methods to screen mutant lines generated by the Zebrafish Mutation Resource under the direction of Dr. Derek Stemple. We will first conduct a primary screen to identify mutants defective in digestive organ lipid uptake, metabolism, transport and storage by feeding fluorescent lipids and lipophilic dyes and assaying their patterns of accumulation in live larvae. We will then conduct secondary screens to comprehensively characterize the phenotypes of identified lipid metabolism and adipose tissue mutants. The overall objective of the proposed research is to identify important genetic modifiers of lipid uptake, transport, and storage in the zebrafish. The rationale is that, once the genetic pathways regulating zebrafish lipid metabolism are known, this information could be translated to humans to initiate new therapeutic approaches to reduce risk of CVD, obesity, IR, and associated disorders by controlling distinct lipid metabolic processes in selected tissues.

Apolipoprotein-B (ApoB) is both a biomarker and a causal mediator of many central hallmarks of metabolic disease, including insulin resistance, fatty liver disease, atherogenesis, endoplasmic reticulum stress, and chronic inflammation. ApoB therefore serves as a useful phenotypic readout for the identification of compounds that engender diverse metabolic benefits. The present proposal will perform a high-throughput screen (HTS) to identify novel ApoB-lowering compounds using an automated robotics platform that enables screening to take place in live larval zebrafish using a genetically encoded chemiluminescent reporter to sensitively detect ApoB levels in individual fish. To accomplish this effort, we have brought together a team of scientists all located at Johns Hopkins University and leaders in their respective fields. Farber has established the zebrafish as a model for studies of vertebrate lipid metabolism, Mumm has created a powerful HTS zebrafish screening platform, Ahima is a world leader in mammalian energy metabolism and Lectka is an established chemist bringing significant expertise in screen hit prioritization to the effort. The HTS will take place in two iterations, with the first iteration screening a ~3,000 compound library of clinically approved compounds so that hits can be rapidly repurposed to treat a host of disease associated with Apob perturbations. The second iteration screening is much larger effort to maximize compound diversity (30,000 compounds) and discover potentially entirely new avenues for treatment. Hits from the both screens will be subjected to a high-content secondary screen that uses an automated imaging platform to monitor effects on disease progression using a panel of transgenic zebrafish carrying fluorescent reporters of several important metabolic disease risk factors. This secondary screen will efficiently classify and prioritize hits from the primary screen and identify the subset of compounds with validated metabolic benefits in live vertebrate organisms that justify further investigation and therapeutic development. Promising compounds from primary and secondary screening will also be validated for activity in mammalian models, including mouse and human cultured cells. The results of these efforts will be the first ever whole animal HTS for ApoB modifiers coupled with a high-content secondary screen that together will enable the rapid identification of compounds to ameliorate many metabolic disease phenotypes, as well as a collection of hits for the development of novel therapies to combat the growing global burden of metabolic disease.

PROJECT SUMMARY Apolipoprotein-B (ApoB) is both a biomarker and a causal mediator of many central hallmarks of metabolic disease, including insulin resistance, fatty liver disease, atherogenesis, endoplasmic reticulum stress, and chronic inflammation. ApoB therefore serves as a useful phenotypic readout for the identification of compounds that engender diverse metabolic benefits. The present proposal will perform a high-throughput screen (HTS) to identify novel ApoB-lowering compounds using an automated robotics platform that enables screening to take place in live larval zebrafish using a genetically encoded chemiluminescent reporter to sensitively detect ApoB levels in individual fish. To accomplish this effort, we have brought together a team of scientists all located at Johns Hopkins University and leaders in their respective fields. Farber has established the zebrafish as a model for studies of vertebrate lipid metabolism, Mumm has created a powerful HTS zebrafish screening platform, Ahima is a world leader in mammalian energy metabolism and Lectka is an established chemist bringing significant expertise in screen hit prioritization to the effort. The HTS will take place in two iterations, with the first iteration screening a ~3,000 compound library of clinically approved compounds so that hits can be rapidly repurposed to treat a host of disease associated with ApoB perturbations. The second iteration screening is much larger effort to maximize compound diversity (10,000 compounds) and discover potentially entirely new avenues for treatment. Hits from the both screens will be subjected to a high-content secondary screen that uses an automated imaging platform to monitor effects on disease progression using a panel of transgenic zebrafish carrying fluorescent reporters of several important metabolic disease risk factors. This secondary screen will efficiently classify and prioritize hits from the primary screen and identify the subset of compounds with validated metabolic benefits in live vertebrate organisms that justify further investigation and therapeutic development. Promising compounds from primary and secondary screening will also be validated for activity in mammalian models, including mouse and human cultured cells. The results of these efforts will be the first ever whole animal HTS for ApoB modifiers coupled with a high-content secondary screen that together will enable the rapid identification of compounds to ameliorate many metabolic disease phenotypes, as well as a collection of hits for the development of novel therapies to combat the growing global burden of metabolic disease.

Apolipoprotein-B (apoB)-containing lipoproteins are both a biomarker and a causal mediator of many central hallmarks of metabolic disease, including insulin resistance, fatty liver disease, atherosclerosis, obesity and metabolic syndrome. Inhibition of microsomal triglyceride transfer protein (MTP), a heterodimeric complex of MTP and protein disulfide isomerase (PDI) subunits, profoundly reduces specifically atherogenic apoB- containing lipoproteins by 50%, but it causes hepatosteatosis and steatorrhea of the intestine. MTP complex transfers different lipids and assists in the production of apoB-containing lipoproteins. Our recent work provides the first evidence that the triglyceride (TG) and phospholipid (PL) transfer functions of MTP can be decoupled and that inhibition of TG transfer activity in zebrafish does not result in steatosis and these fish grow normally like wild-type fish. We hypothesize that atomic level details about these two lipid transfer domains may pave the way for selective pharmacological inhibition of TG transfer to lower plasma lipids without causing the adverse effects of cellular lipid retention. The fundamental question we are asking is: ?how MTP distinguishes different lipid ligands and what are the consequences of inhibiting TG transfer activity?? Aim 1: Characterize the different lipid-binding sites in MTP: We will solve MTP structures with different lipid ligands to obtain atomic level details. Mutational analysis will elucidate amino acid residues critical for binding of specific lipids. Aim 2: Identify conformational changes in MTP and PDI subunits during lipid transfer, and different PDI family members that interact with MTP subunit: We hypothesize that conformational changes in both the MTP and PDI subunits occur to accommodate different lipids. We will perform site-directed mutagenesis in the flexible loop region of MTP and a? domain of PDI to dissect out the mechanisms for this. Although PDI is obligatory for MTP activity, the specificity of different PDI paralogs is unknown. We will test the hypothesis that other PDI family members interact with the MTP subunit and these interactions have physiological consequences. Aim 3: Assess the biological consequences of abolishing TG transfer activity of MTP: After identifying further mutations that abolish TG transfer, we will determine whether these mutants support apoB secretion in cells, lower plasma lipids in mice, and sustain normal fish growth. The proposed studies will provide novel information about 1) the domains and amino acids in the transfer of different lipids by MTP; 2) conformational changes that occur during transfer of different lipids; and 3) biochemical, physiological and organismal consequences of mutating these critical residues. This new knowledge will be invaluable, in the future, to develop novel and TG transfer specific inhibitors of MTP.