Greg L. Harris

TNFα receptor expression in rat cardiac myocytes: TNFα inhibition of L-type Ca2+ current and Ca2+ transients

Tumor necrosis factor‐α (TNFα) is a potentially powerful anti‐neoplastic agent; however, its therapeutic usefulness is limited by its cardiotoxic and negative inotropic effects. Accordingly, studies were undertaken to gain a better understanding of the mechanisms of TNFα‐mediated cardiodepression. Single cell RT‐PCR, [125I]TNFα ligand binding and Western immunoblotting experiments demonstrated that rat cardiac cells predominantly express type I TNFα receptors (TNFRI or p60). TNFα inhibited cardiac L‐type Ca2+ channel current (I Ca) and contractile Ca2+ transients. Thus, it is possible that the negative inotropic effects of TNFα are the result of TNFRI‐mediated blockade of cardiac excitation‐contraction coupling.

p62, Ref(2)P and ubiquitinated proteins are conserved markers of neuronal aging, aggregate formation and progressive autophagic defects

Suppression of macroautophagy, due to mutations or through processes linked to aging, results in the accumulation of cytoplasmic substrates that are normally eliminated by the pathway. This is a significant problem in long-lived cells like neurons, where pathway defects can result in the accumulation of aggregates containing ubiquitinated proteins. The p62/Ref(2)P family of proteins is involved in the autophagic clearance of cytoplasmic protein bodies or sequestosomes. These unique structures are closely associated with protein inclusions containing ubiquitin as well as key components of the autophagy pathway. In this study we show that detergent fractionation followed by western blot analysis of insoluble ubiquitinated proteins (IUP), mammalian p62 and its Drosophila homologue, Ref(2)P can be used to quantitatively assess the activity level of aggregate clearance (aggrephagy) in complex tissues. Using this technique we show that genetic or age-dependent changes that modify the long-term enhancement or suppression of aggrephagy can be identified. Moreover, using the Drosophila model system this method can be used to establish autophagy-dependent protein clearance profiles that are occurring under a wide range of physiological conditions including developmental, fasting and altered metabolic pathways. This technique can also be used to examine proteopathies that are associated with human disorders such as frontotemporal dementia, Huntington and Alzheimer disease. Our findings indicate that measuring IUP profiles together with an assessment of p62/Ref(2)P proteins can be used as a screening or diagnostic tool to characterize genetic and age-dependent factors that alter the long-term function of autophagy and the clearance of protein aggregates occurring within complex tissues and cells.

Sply regulation of sphingolipid signaling molecules is essential for Drosophila development.

Sphingosine-1-phosphate is a sphingolipid metabolite that regulates cell proliferation, migration and apoptosis through specific signaling pathways. Sphingosine-1-phosphate lyase catalyzes the conversion of sphingosine-1-phosphate to ethanolamine phosphate and a fatty aldehyde. We report the cloning of the Drosophila sphingosine-1-phosphate lyase gene (Sply) and demonstrate its importance for adult muscle development and integrity, reproduction and larval viability. Sply expression is temporally regulated, with onset of expression during mid-embryogenesis. Sply null mutants accumulate both phosphorylated and unphosphorylated sphingoid bases and exhibit semi-lethality, increased apoptosis in developing embryos, diminished egg-laying, and gross pattern abnormalities in dorsal longitudinal flight muscles. These defects are corrected by restoring Sply expression or by introduction of a suppressor mutation that diminishes sphingolipid synthesis and accumulation of sphingolipid intermediates. This is the first demonstration of novel and complex developmental pathologies directly linked to a disruption of sphingolipid catabolism in metazoans.

Characterization of the drosophila sphingosine kinases and requirement for Sk2 in normal reproductive function

Sphingosine kinase is a highly conserved enzyme that catalyzes the synthesis of sphingosine 1-phosphate and reduces cellular levels of sphingosine and ceramide. Although ceramide is pro-apoptotic and sphingosine is generally growth-inhibitory, sphingosine 1-phosphate signaling promotes cell proliferation, survival, and migration. Sphingosine kinase is thus in a strategic position to regulate important cell fate decisions which may contribute to normal animal development. To facilitate studies examining the potential role of sphingosine kinase and long chain base metabolism in Drosophila development, we characterized two putative Drosophila sphingosine kinase genes, Sk1 and Sk2. Both genes functionally and biochemically complement a yeast sphingosine kinase mutant, express predominantly cytosolic activities, and are capable of phosphorylating a range of endogenous and non-endogenous sphingoid base substrates. The two genes demonstrate overlapping but distinct temporal and spatial expression patterns in the Drosophila embryo, and timing of expression is consistent with observed changes in long chain base levels throughout development. A null Sk2 transposon insertion mutant demonstrated elevated long chain base levels, impaired flight performance, and diminished ovulation. This is the first reported mutation of a sphingosine kinase in an animal model; the associated phenotypes indicate that Sk1 and Sk2 are not redundant in biological function and that sphingosine kinase is essential for diverse physiological functions in this organism.

Identification and characterization by electrospray mass spectrometry of endogenous Drosophila sphingadienes.

Sphingolipids comprise a complex group of lipids concentrated in membrane rafts and whose metabolites function as signaling molecules. Sphingolipids are conserved in Drosophila, in which their tight regulation is required for proper development and tissue integrity. In this study, we identified a new family of Drosophila sphingolipids containing two double bonds in the long chain base (LCB). The lipids were found at low levels in wild-type flies and accumulated markedly in Drosophila Sply mutants, which do not express sphingosine-1-phosphate lyase and are defective in sphingolipid catabolism. To determine the identity of the unknown lipids, purified whole fly lipid extracts were separated on a C18-HPLC column and analyzed using electrospray mass spectrometry. The lipids contain a LCB of either 14 or 16 carbons with conjugated double bonds at C4,6. The Δ4,6-sphingadienes were found as free LCBs, as phosphorylated LCBs, and as the sphingoid base in ceramides. The temporal and spatial accumulation of Δ4,6-sphingadienes in Sply mutants suggests that these lipids may contribute to the muscle degeneration observed in these flies.

The K+ Channel Gene Ether a Go-Go Is Required for the Transduction of a Subset of Odorants in Adult Drosophila melanogaster

The functional identity of an olfactory receptor neuron is determined in part by its repertoire of responses to odorants. As an approach toward understanding the contributions of particular conductances to olfactory neuron excitability and odor discrimination, we have investigated the role of the putative cyclic nucleotide-modulated K+ channel subunit encoded by the ether a go-go (eag) gene in odorant responsiveness in Drosophila melanogaster. Four independent mutant eag alleles exhibited reduced antennal sensitivity to a subset of nine odorants, all having short aliphatic side chains: ethyl butyrate (EB), propionic acid, 2-butanone, and ethyl acetate. Significantly fewer eag antennal neurons responded to EB compared with control neurons; the proportion sensitive to 2-heptanone was similar to controls. Two aspects of the character of EB-induced excitability were affected by mutations ineag. First, fewer EB-induced inhibitory responses were observed in eag mutants, and second, fewer excitatory odorant responses dependent on extracellular Ca2+were observed. Furthermore, modulation of neuronal excitability by membrane-permeant cyclic nucleotide analogs was largelyeag dependent. Focal application of high K+ saline to sensillae altered the excitability of the majority of neurons from wild-type but not eagantennae, suggesting that Eag may have a dendritic localization.

In Vitro and In Vivo Antagonism of a G Protein-Coupled Receptor (S1P3) with a Novel Blocking Monoclonal Antibody

Background S1P3 is a lipid-activated G protein-couple receptor (GPCR) that has been implicated in the pathological processes of a number of diseases, including sepsis and cancer. Currently, there are no available high-affinity, subtype-selective drug compounds that can block activation of S1P3. We have developed a monoclonal antibody (7H9) that specifically recognizes S1P3 and acts as a functional antagonist. Methodology/Principal Findings Specific binding of 7H9 was demonstrated by immunocytochemistry using cells that over-express individual members of the S1P receptor family. We show, in vitro, that 7H9 can inhibit the activation of S1P3-mediated cellular processes, including arrestin translocation, receptor internalization, adenylate cyclase inhibiton, and calcium mobilization. We also demonstrate that 7H9 blocks activation of S1P3 in vivo, 1) by preventing lethality due to systemic inflammation, and 2) by altering the progression of breast tumor xenografts. Conclusions/Significance We have developed the first-reported monoclonal antibody that selectively recognizes a lipid-activated GPCR and blocks functional activity. In addition to serving as a lead drug compound for the treatment of sepsis and breast cancer, it also provides proof of concept for the generation of novel GPCR-specific therapeutic antibodies.

VOLTAGE-ACTIVATED AND ODOR-MODULATED CONDUCTANCES IN OLFACTORY NEURONS OF DROSOPHILA MELANOGASTER

Voltage-activated currents and odor-modulated conductances were studied in cells in semi-intact Drosophila third antennal segments (the main olfactory organ) using patch-clamp techniques. All neurons expressed outward currents, and most expressed labile fast transient inward currents with kinetics similar to Na+ currents in other systems. Action potentials were detected as bipolar capacitative current transients in cell-attached or loose patches from the soma of both odor-sensitive (97%) and insensitive neurons. A mixture of odorants from five chemical classes caused an increase (approximately 70%), decrease (approximately 10%), or no effect on firing frequency in pharate adult neurons. The development of chemosensitivity was examined and odor-induced changes in action potential firing frequency were recorded in pupal antennal neurons as early as P8, a stage after completion of sensillar development. The character of odor-induced responses was more profound and complex later in development; small, tonic increases in firing frequency were observed at pupal stages P8 through P11 (ii), while in older pupae and young adults approximately 25% of the increased responses were phasic-tonic. The apical dendrite was the site of odor modulation in approximately 90% and 100% of responsive adult and early pupal neurons, respectively. Whole-cell recordings revealed that apparent nonselective cation and chloride conductances were modulated by a mixture of odorants in separate antennal neurons.

Close head‐to‐head juxtaposition of genes favors their coordinate regulation in Drosophila melanogaster

This report identifies a large number of gene‐pairs in Drosophila melanogaster that share a common upstream region. 877 gene‐pairs (∼12% of the genome) are separated by less than 350 bp in a head‐to‐head orientation. This positional relationship is more highly favored in flies than in other organisms. These gene pairs have a higher correlation of expression than similarly spaced genes that have head‐to‐tail or tail‐to‐tail orientations. Thus, the positional arrangement of genes appears to play a significant role in coordinating relative expression patterns and may provide clues for identifying the functions of unknown genes.

Identification of Sphingolipid Metabolites That Induce Obesity via Misregulation of Appetite, Caloric Intake and Fat Storage in Drosophila

Obesity is defined by excessive lipid accumulation. However, the active mechanistic roles that lipids play in its progression are not understood. Accumulation of ceramide, the metabolic hub of sphingolipid metabolism, has been associated with metabolic syndrome and obesity in humans and model systems. Here, we use Drosophila genetic manipulations to cause accumulation or depletion of ceramide and sphingosine-1-phosphate (S1P) intermediates. Sphingolipidomic profiles were characterized across mutants for various sphingolipid metabolic genes using liquid chromatography electrospray ionization tandem mass spectroscopy. Biochemical assays and microscopy were used to assess classic hallmarks of obesity including elevated fat stores, increased body weight, resistance to starvation induced death, increased adiposity, and fat cell hypertrophy. Multiple behavioral assays were used to assess appetite, caloric intake, meal size and meal frequency. Additionally, we utilized DNA microarrays to profile differential gene expression between these flies, which mapped to changes in lipid metabolic pathways. Our results show that accumulation of ceramides is sufficient to induce obesity phenotypes by two distinct mechanisms: 1) Dihydroceramide (C14:0) and ceramide diene (C14:2) accumulation lowered fat store mobilization by reducing adipokinetic hormone- producing cell functionality and 2) Modulating the S1P: ceramide (C14:1) ratio suppressed postprandial satiety via the hindgut-specific neuropeptide like receptor dNepYr, resulting in caloric intake-dependent obesity.