Highly Unsaturated Fatty Acid Synthesis in Atlantic Salmon: Characterization of ELOVL5- and ELOVL2-like Elongases

Fish species vary in their capacity to biosynthesize the n-3 long-chain polyunsaturated fatty acids (LC-PUFA) eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids that are crucial to the health of higher vertebrates. The synthesis of LC-PUFA involves enzyme-mediated fatty acyl desaturation and elongation. Previously, a cDNA for an elongase, now termed elovl5a, had been cloned from Atlantic salmon. Here we report on the cloning of two new elongase cDNAs: a second elovl5b elongase, corresponding to a 294 aa protein, and an elovl2-like elongase, coding for a 287 aa protein, characterized for the first time in a non-mammalian vertebrate. Heterologous expression in yeast showed that the salmon Elovl5b elongated C18 and C20 PUFA, with low activity towards C22, while Elovl2 elongated C20 and C22 PUFA with lower activity towards C18 PUFA. All three transcripts showed predominant expression in the intestine and liver, followed by the brain. Elongase expression showed differential nutritional regulation. Levels of elovl5b and particularly of elovl2, but not of elovl5a, transcripts were significantly increased in liver of salmon fed vegetable oils (VO) compared to fish fed fish oil (FO). Intestinal expression showed a similar pattern. Phylogenetic comparisons indicate that, in contrast to salmon and zebrafish, Acanthopterygian fish species lack elovl2 which is consistent with their neglible ability to biosynthesise LC-PUFA and to adapt to VO dietary inclusion, compared to predominantly freshwater salmonids. Thus the presence of elovl2 in salmon explains the ability of this species to biosynthesise LC-HUFA and may provide a biotechnological tool to produce enhanced levels of LC-PUFA, particularly DHA, in transgenic organisms

Functional Desaturase Fads1 (Δ5) and Fads2 (Δ6) Orthologues Evolved before the Origin of Jawed Vertebrates

Long-chain polyunsaturated fatty acids (LC-PUFAs) such as arachidonic (ARA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids are essential components of biomembranes, particularly in neural tissues. Endogenous synthesis of ARA, EPA and DHA occurs from precursor dietary essential fatty acids such as linoleic and α-linolenic acid through elongation and Δ5 and Δ6 desaturations. With respect to desaturation activities some noteworthy differences have been noted in vertebrate classes. In mammals, the Δ5 activity is allocated to the Fads1 gene, while Fads2 is a Δ6 desaturase. In contrast, teleosts show distinct combinations of desaturase activities (e.g. bifunctional or separate Δ5 and Δ6 desaturases) apparently allocated to Fads2-type genes. To determine the timing of Fads1-Δ5 and Fads2-Δ6 evolution in vertebrates we used a combination of comparative and functional genomics with the analysis of key phylogenetic species. Our data show that Fads1 and Fads2 genes with Δ5 and Δ6 activities respectively, evolved before gnathostome radiation, since the catshark Scyliorhinus canicula has functional orthologues of both gene families. Consequently, the loss of Fads1 in teleosts is a secondary episode, while the existence of Δ5 activities in the same group most likely occurred through independent mutations into Fads2 type genes. Unexpectedly, we also establish that events of Fads1 gene expansion have taken place in birds and reptiles. Finally, a fourth Fads gene (Fads4) was found with an exclusive occurrence in mammalian genomes. Our findings enlighten the history of a crucially important gene family in vertebrate fatty acid metabolism and physiology and provide an explanation of how observed lineage-specific gene duplications, losses and diversifications might be linked to habitat-specific food web structures in different environments and over geological timescales

Investigation of highly unsaturated fatty acid metabolism in the Asian sea bass, Lates calcarifer

Lates calcarifer, commonly known as the Asian sea bass or barramundi, is an interesting species that has great aquaculture potential in Asia including Malaysia and also Australia. We have investigated essential fatty acid metabolism in this species, focusing on the endogenous highly unsaturated fatty acid (HUFA) synthesis pathway using both biochemical and molecular biological approaches. Fatty acyl desaturase (Fad) and elongase (Elovl) cDNAs were cloned and functional characterization identified them as ∆6 Fad and Elovl5 elongase enzymes, respectively. The ∆6 Fad was equally active towards 18:3n-3 and 18:2n-6, and Elovl5 exhibited elongation activity for C18-20 and C20-22 elongation and a trace of C22-24 activity. The tissue profile of gene expression for ∆6 fad and elovl5 genes, showed brain to have the highest expression of both genes compared to all other tissues. The results of tissue fatty acid analysis showed that the brain contained more docosahexaenoic acid (DHA, 22:6n-3) than flesh, liver and intestine. The HUFA synthesis activity in isolated hepatocytes and enterocytes using [1-14C]18:3n-3 as substrate was very low with the only desaturated product detected being 18:4n-3. These findings indicate that L. calcarifer display an essential fatty acid pattern similar to other marine fish in that they appear unable to synthesize HUFA from C18 substrates. High expression of ∆6 fad and elovl5 genes in brain may indicate a role for these enzymes in maintaining high DHA levels in neural tissues through conversion of 20:5n-3

Genetic Determinants of Phosphate Response in Drosophila

Phosphate is required for many important cellular processes and having too little phosphate or too much can cause disease and reduce life span in humans. However, the mechanisms underlying homeostatic control of extracellular phosphate levels and cellular effects of phosphate are poorly understood. Here, we establish Drosophila melanogaster as a model system for the study of phosphate effects. We found that Drosophila larval development depends on the availability of phosphate in the medium. Conversely, life span is reduced when adult flies are cultured on high phosphate medium or when hemolymph phosphate is increased in flies with impaired Malpighian tubules. In addition, RNAi-mediated inhibition of MAPK-signaling by knockdown of Ras85D, phl/D-Raf or Dsor1/MEK affects larval development, adult life span and hemolymph phosphate, suggesting that some in vivo effects involve activation of this signaling pathway by phosphate. To identify novel genetic determinants of phosphate responses, we used Drosophila hemocyte-like cultured cells (S2R+) to perform a genome-wide RNAi screen using MAPK activation as the readout. We identified a number of candidate genes potentially important for the cellular response to phosphate. Evaluation of 51 genes in live flies revealed some that affect larval development, adult life span and hemolymph phosphate levels

Plakophilin-3 Is Required for Late Embryonic Amphibian Development, Exhibiting Roles in Ectodermal and Neural Tissues

The p120-catenin family has undergone a significant expansion during the evolution of vertebrates, resulting in varied functions that have yet to be discerned or fully characterized. Likewise, members of the plakophilins, a related catenin subfamily, are found throughout the cell with little known about their functions outside the desmosomal plaque. While the plakophilin-3 (Pkp3) knockout mouse resulted in skin defects, we find larger, including lethal effects following its depletion in Xenopus. Pkp3, unlike some other characterized catenins in amphibians, does not have significant maternal deposits of mRNA. However, during embryogenesis, two Pkp3 protein products whose temporal expression is partially complimentary become expressed. Only the smaller of these products is found in adult Xenopus tissues, with an expression pattern exhibiting distinctions as well as overlaps with those observed in mammalian studies. We determined that Xenopus Pkp3 depletion causes a skin fragility phenotype in keeping with the mouse knockout, but more novel, Xenopus tailbud embryos are hyposensitive to touch even in embryos lacking outward discernable phenotypes, and we additionally resolved disruptions in certain peripheral neural structures, altered establishment and migration of neural crest, and defects in ectodermal multiciliated cells. The use of two distinct morpholinos, as well as rescue approaches, indicated the specificity of these effects. Our results point to the requirement of Pkp3 in amphibian embryogenesis, with functional roles in a number of tissue types

25th Annual Computational Neuroscience Meeting: CNS-2016

Abstracts of the 25th Annual Computational Neuroscience Meeting: CNS-2016 Seogwipo City, Jeju-do, South Korea. 2–7 July 201