Generation and characterization of new alleles of quiver (qvr) that encodes an extracellular modulator of the Shaker potassium channel.

Our earlier genetic screen uncovered a paraquat-sensitive leg-shaking mutant quiver1 (qvr1), whose gene product interacts with the Shaker (Sh) K+ channel. We also mapped the qvr locus to EY04063 and noticed altered day-night activity patterns in these mutants. Such circadian behavioral defects were independently reported by another group, who employed the qvr1 allele we supplied them, and attributed the extreme restless phenotype of EY04063 to the qvr gene. However, their report adopted a new noncanonical gene name sleepless (sss) for qvr. In addition to qvr1 and qvrEY, our continuous effort since the early 2000s generated a number of novel recessive qvr alleles, including ethyl methanesulfonate (EMS)-induced mutations qvr2 and qvr3, and P-element excision lines qvrip6 (imprecise jumpout), qvrrv7, and qvrrv9 (revertants) derived from qvrEY. Distinct from the original intron-located qvr1 allele that generates abnormal-sized mRNAs, qvr2, and qvr3 had their lesion sites in exons 6 and 7, respectively, producing nearly normal-sized mRNA products. A set of RNA-editing sites are nearby the lesion sites of qvr3 and qvrEY on exon 7. Except for the revertants, all qvr alleles display a clear ether-induced leg-shaking phenotype just like Sh, and weakened climbing abilities to varying degrees. Unlike Sh, all shaking qvr alleles (except for qvrf01257) displayed a unique activity-dependent enhancement in excitatory junction potentials (EJPs) at larval neuromuscular junctions (NMJs) at very low stimulus frequencies, with qvrEY displaying the largest EJP and more significant NMJ overgrowth than other alleles. Our detailed characterization of a collection of qvr alleles helps to establish links between novel molecular lesions and different behavioral and physiological consequences, revealing how modifications of the qvr gene lead to a wide spectrum of phenotypes, including neuromuscular hyperexcitability, defective motor ability and activity-rest cycles.

Expression of zinc-deficient human superoxide dismutase in Drosophila neurons produces a locomotor defect linked to mitochondrial dysfunction.

More than 130 different mutations in the Cu/Zn superoxide dismutase (SOD1) gene have been associated with amyotrophic lateral sclerosis but the mechanism of this toxicity remains controversial. To gain insight into the importance of the zinc site in the pathogenesis of SOD1 in vivo, we generated a Drosophila model with transgenic expression of a zinc-deficient human SOD1. Expression of zinc-deficient SOD1 in Drosophila resulted in a progressive movement defect with associated mitochondrial cristae vacuolization and reductions in adenosine triphosphate (ATP) levels. Furthermore, these flies are sensitized to mitochondrial toxins, paraquat, and zinc. Importantly, we show that the zinc-deficient SOD1-induced motor defect can be ameliorated by supplementing the endogenous fly respiratory chain machinery with the single-subunit NADH-ubiquinone oxidoreductase from yeast (NADH is nicotinamide adenine dinucleotide, reduced form.). These results demonstrate that zinc-deficient SOD1 is neurotoxic in vivo and suggest that mitochondrial dysfunction plays a critical role in this toxicity. The robust behavioral, pathological, and biochemical phenotypes conferred by zinc-deficient SOD1 in Drosophila have general implications for the role of the zinc ion in familial and sporadic amyotrophic lateral sclerosis.

Overexpression of metal-responsive transcription factor (MTF-1) in Drosophila melanogaster ameliorates life-span reductions associated with oxidative stress and metal toxicity.

Heavy metals are essential components of many biological processes but are toxic at high concentrations. Our results illustrate that when metal homeostasis is compromised by a mutation in the metal-responsive transcription factor (MTF-1), the life-span is shortened. In contrast, MTF-1 overexpression results in resistant flies with prolonged longevity on iron or cadmium-supplemented media but shortened life-span on zinc-supplemented medium. This effect was mediated by the overexpression of MTF-1 in specific tissues, such as the gut, hemocytes and in particular in neurons, indicating that these tissues are particularly sensitive to the perturbance of metal homeostasis. Further, MTF-1 overexpression in a neuron-specific manner protects flies against hyperoxia and prolongs the life-span of Cu/Zn superoxide dismutase-deficient flies, suggesting the presence of a common mechanism for protection against both oxidative stress and metal toxicity. Finally, normal life-span is extended up to 40% upon MTF-1 overexpression in either the peripheral nervous system or motorneurons. These results document the tissue-specific import of heavy metal toxicity and oxidative damage in aging and life-span determination.

Mutations of the withered (whd) gene in Drosophila melanogaster confer hypersensitivity to oxidative stress and are lesions of the carnitine palmitoyltransferase I (CPT I) gene.

Since some oxygen defense mutants of Drosophila melanogaster exhibit a crinkled wing phenotype, a screen was performed on strains bearing mutant alleles conferring a visible wing phenotype to determine whether any were hypersensitive to oxidative stress. One mutant, withered (whd), was found to be sensitive to both dietary paraquat and hyperoxia. New alleles of whd were induced on a defined genetic background and strains carrying these alleles were also found to be sensitive to oxidative stress. To identify the product of the whd gene we used a sequence-based positional candidate approach and by this method we determined that whd encodes carnitine palmitoyltransferase I (CPT I), an enzyme of the outer mitochondrial membrane that is required for the import of long-chain fatty acids into the mitochondria for beta-oxidation. Although this function is not vital under laboratory conditions, whd adults were found to be highly sensitive to starvation and to heavy metal toxicity relative to controls. This work uncovers a novel relationship between fatty acid metabolism and reactive oxygen metabolism. Further, these results in conjunction with past research on whd and on mammalian CPT I support the hypothesis that CPT I serves a vital function in the response to thymine supplementation.

Cloning of a second form of activin-betaA cDNA and regulation of activin-betaA subunits and activin type II receptor mRNA expression by gonadotropin in the zebrafish ovary.

Activins are dimeric proteins consisting of two inhibin beta subunits. Homo- and hetero-dimerizations of two isoforms of beta subunits, betaA and betaB, produce three forms of activins, activin-A, -B, and -AB. Recent studies have suggested that activin-A mediates gonadotropin-induced oocyte maturation in the zebrafish. To further understand the physiological role of activin-A in the zebrafish ovary, we have cloned cDNAs for a second isoform of the activin-betaA subunit and the activin type IIA (ActRIIA) receptor and determined their regulation by gonadotropin. Two sequences were obtained during the cloning of activin-betaA subunit, both of which showed high identity to betaA subunits of other species, and were therefore designated as isoform 1 and 2. Real-time PCR quantification was used to measure mRNA levels of activin-betaA1 and -betaA2, as well as two type II receptors, ActRIIA and ActRIIB, in the zebrafish ovary. Activin-betaA1 mRNA levels in stages III and IV follicles were similar and higher than those in stage II while high activin-betaA2 mRNA levels were only found in stage IV follicles. Highest levels of mRNA expression were detected in small and large stage III follicles for ActRIIA and ActRIIB, respectively. Treatment with human chorionic gonadotropin induced dose- and time-dependent increases in mRNA levels of activin-betaA1 and -betaA, as well as ActRIIA and ActRIIB. These findings further support the involvement of the activin signaling cascade in gonadotropin-regulated gonadal activities.