Knock-Down of Cathepsin D Affects the Retinal Pigment Epithelium, Impairs Swim-Bladder Ontogenesis and Causes Premature Death in Zebrafish

The lysosomal aspartic protease Cathepsin D (CD) is ubiquitously expressed in eukaryotic organisms. CD activity is essential to accomplish the acid-dependent extensive or partial proteolysis of protein substrates within endosomal and lysosomal compartments therein delivered via endocytosis, phagocytosis or autophagocytosis. CD may also act at physiological pH on small-size substrates in the cytosol and in the extracellular milieu. Mouse and fruit fly CD knock-out models have highlighted the multi-pathophysiological roles of CD in tissue homeostasis and organ development. Here we report the first phenotypic description of the lack of CD expression during zebrafish (Danio rerio) development obtained by morpholino-mediated knock-down of CD mRNA. Since the un-fertilized eggs were shown to be supplied with maternal CD mRNA, only a morpholino targeting a sequence containing the starting ATG codon was effective. The main phenotypic alterations produced by CD knock-down in zebrafish were: 1. abnormal development of the eye and of retinal pigment epithelium; 2. absence of the swim-bladder; 3. skin hyper-pigmentation; 4. reduced growth and premature death. Rescue experiments confirmed the involvement of CD in the developmental processes leading to these phenotypic alterations. Our findings add to the list of CD functions in organ development and patho-physiology in vertebrates

Determination of ubiquinone and related metabolites in zebrafish embryos by LC-HRMS.

Introduction Characterization of lipidome is an emerging topic in metabolomics and the contribution of HPLC-HRMS could be an important tool in the elucidation of biochemical pathways. One goal of this work is to provide a rapid, selective and sensitive method to measure ubiquinone and related lipids concentration in zebrafish embryos and to apply it to metabolism studies. Subsequently untargeted analysis of related lipophilic compounds becomes achievable. Ubiquinone (also known as coenzyme Q10) plays an essential role in the mitochondria electron-transport chain. It is a interesting molecule shown to play an important antioxidant role in the cardiovascular system and it is the only endogenously synthesized lipid-soluble antioxidant. Zebrafish (Danio rerio) is an established model for studying toxicology and understanding human diseases. Methods Embryos and adult fishes were raised and maintained under standard laboratory conditions. Biological samples were extracted by LLE with various organic solvents. HPLC-HRMS analyses were accomplished on a Dionex Ultimate 3000 LC system coupled with a LTQ-Orbitrap instrument, with ESI and APCI interfaces. C4, C8 and C18 RP columns were tested for separation. Here we investigate the ionization modes, the ex vivo analytical sensitivity and the fragmentation mechanisms of ubiquinone and related compounds. The developed methodology will be applied to study samples of zebrafish embryos. Preliminary data In the first steps of method development we optimized the chromatographic separation of highly lipophilic ubiquinone related compounds on RP-HPLC columns and studied the sensitivity of different ionization modes (positive vs. negative ion mode, APCI vs. ESI, proton vs ammonium vs. sodium vs. lithium adducts formation). A fragmentation study of the analytes was also done evidencing a double neutral loss of formaldehyde as major pathway. A full validation study was then completed to make possible quantitative determination on biological samples. Accuracy, precision, LLOQ, linearity, extraction recovery were evaluated. Then to confirm the high similarity between zebrafish and higher vertebrates at the cellular and physiological levels we determined the variation of concentration of coenzyme Q10 at various zebrafish growth steps and compared it with similar metabolites as coenzymes with shorter isoprene chain (Q9 to Q6). Finally a study to characterize the untargeted lipidome of embryo samples is ongoing to resolve the complex mixture of compounds giving mono-charged ions in the range 700-1000 m/z. Novel aspects Rapid quantification of ubiquinone and untargeted lipidomic analysis in ex vivo zebrafish samples for biochemical studies

Ubiad1 is an antioxidant enzyme that regulates eNOS activity by CoQ10 synthesis

Protection against oxidative damage caused by excessive reactive oxygen species (ROS) by an antioxidant network is essential for the health of tissues, especially in the cardiovascular system. Here, we identified a gene with important antioxidant features by analyzing a null allele of zebrafish ubiad1, called barolo (bar). bar mutants show specific cardiovascular failure due to oxidative stress and ROS-mediated cellular damage. Human UBIAD1 is a nonmitochondrial prenyltransferase that synthesizes CoQ10 in the Golgi membrane compartment. Loss of UBIAD1 reduces the cytosolic pool of the antioxidant CoQ10 and leads to ROS-mediated lipid peroxidation in vascular cells. Surprisingly, inhibition of eNOS prevents Ubiad1-dependent cardiovascular oxidative damage, suggesting a crucial role for this enzyme and nonmitochondrial CoQ10 in NO signaling. These findings identify UBIAD1 as a nonmitochondrial CoQ10-forming enzyme with specific cardiovascular protective function via the modulation of eNOS activity

Ubiad1 is an antioxidant enzyme that regulates eNOS activity by CoQ10 synthesis

Protection against oxidative damage caused by excessive reactive oxygen species (ROS) by an antioxidant network is essential for the health of tissues, especially in the cardiovascular system. Here, we identified a gene with important antioxidant features by analyzing a null allele of zebrafish ubiad1, called barolo (bar). bar mutants show specific cardiovascular failure due to oxidative stress and ROS-mediated cellular damage. Human UBIAD1 is a nonmitochondrial prenyltransferase that synthesizes CoQ10 in the Golgi membrane compartment. Loss of UBIAD1 reduces the cytosolic pool of the antioxidant CoQ10 and leads to ROS-mediated lipid peroxidation in vascular cells. Surprisingly, inhibition of eNOS prevents Ubiad1-dependent cardiovascular oxidative damage, suggesting a crucial role for this enzyme and nonmitochondrial CoQ10 in NO signaling. These findings identify UBIAD1 as a nonmitochondrial CoQ10-forming enzyme with specific cardiovascular protective function via the modulation of eNOS activity