Neuroprotection and repair of 3'-blocking DNA ends by glaikit (gkt) encoding Drosophila tyrosyl-DNA phosphodiesterase 1 (TDP1).

Tyrosyl-DNA phosphodiesterase (TDP1) is a phylogenetically conserved enzyme critical for the removal of blocking lesions at the 3' ends of DNA or RNA. This study analyzes the Drosophila TDP1 gene ortholog glaikit (gkt) and its possible role(s) in the repair of endogenous DNA lesions and neuroprotection. To do so, we studied a homozygous PiggyBac insertion (c03958) that disrupts the 5' UTR of gkt. Protein extracts of c03958 flies were defective in hydrolyzing 3'-DNA-tyrosyl residues, demonstrating that gkt is the Drosophila TDP1. Although the mutant is generally healthy and fertile, females exhibit reduced lifespan and diminished climbing ability. This phenotype was rescued by neuronal expression of TDP1. In addition, when c03958 larvae were exposed to bleomycin, an agent that produces oxidative DNA damage, or topoisomerase I-targeted drugs (camptothecin and a noncamptothecin indenoisoquinoline derivative, LMP-776), survivors displayed rough eye patches, which were rescued by neuronal expression of TDP1. Our study establishes that gkt is the Drosophila TDP1 gene, and that it is critical for neuroprotection, normal longevity, and repair of damaged DNA.

Drosophila ryanodine receptors mediate general anesthesia by halothane.

BACKGROUND: Although in vitro studies have identified numerous possible targets, the molecules that mediate the in vivo effects of volatile anesthetics remain largely unknown. The mammalian ryanodine receptor (Ryr) is a known halothane target, and the authors hypothesized that it has a central role in anesthesia. METHODS: Gene function of the Drosophila Ryr (dRyr) was manipulated in the whole body or in specific tissues using a collection of mutants and transgenes, and responses to halothane were measured with a reactive climbing assay. Cellular responses to halothane were studied using Ca imaging and patch clamp electrophysiology. RESULTS: Halothane potency strongly correlates with dRyr gene copy number, and missense mutations in regions known to be functionally important in the mammalian Ryrs gene cause dominant hypersensitivity. Tissue-specific manipulation of dRyr shows that expression in neurons and glia, but not muscle, mediates halothane sensitivity. In cultured cells, halothane-induced Ca efflux is strictly dRyr-dependent, suggesting a close interaction between halothane and dRyr. Ca imaging and electrophysiology of Drosophila central neurons reveal halothane-induced Ca flux that is altered in dRyr mutants and correlates with strong hyperpolarization. CONCLUSIONS: In Drosophila, neurally expressed dRyr mediates a substantial proportion of the anesthetic effects of halothane in vivo, is potently activated by halothane in vitro, and activates an inhibitory conductance. The authors' results provide support for Ryr as an important mediator of immobilization by volatile anesthetics.

A putative cation channel and its novel regulator: cross-species conservation of effects on general anesthesia.

Volatile anesthetics like halothane and enflurane are of interest to clinicians and neuroscientists because of their ability to preferentially disrupt higher functions that make up the conscious state. All volatiles were once thought to act identically; if so, they should be affected equally by genetic variants. However, mutations in two distinct genes, one in Caenorhabditis and one in Drosophila, have been reported to produce much larger effects on the response to halothane than enflurane [1, 2]. To see whether this anesthesia signature is adventitious or fundamental, we have identified orthologs of each gene and determined the mutant phenotype within each species. The fly gene, narrow abdomen (na), encodes a putative ion channel whose sequence places it in a unique family; the nematode gene, unc-79, is identified here as encoding a large cytosolic protein that lacks obvious motifs. In Caenorhabditis, mutations that inactivate both of the na orthologs produce an Unc-79 phenotype; in Drosophila, mutations that inactivate the unc-79 ortholog produce an na phenotype. In each organism, studies of double mutants place the genes in the same pathway, and biochemical studies show that proteins of the UNC-79 family control NA protein levels by a posttranscriptional mechanism. Thus, the anesthetic signature reflects an evolutionarily conserved role for the na orthologs, implying its intimate involvement in drug action.

Visual mutations reveal opposing effects of illumination on arousal in Drosophila.

The effect of illumination on alertness can be assessed by comparing the efficacy of an anesthetic under light vs. dark conditions. Results from such tests on wild-type flies and visual mutants demonstrate that, surprisingly, light has both positive and negative influences on arousal. These dual effects may explain aspects of the fly's daily activity and have potential clinical implications.

Genetic effects in Drosophila on the potency of diverse general anesthetics: a distinctive pattern of altered sensitivity.

Mutations that influence the sensitivity of an organism to a volatile general anesthetic can be divided into two classes. In one, sensitivity to all other volatile agents is affected to a similar degree. Although this class may contain mutations of interest for understanding anesthesia, it is also likely to contain mutations that merely alter general health. In the second class, mutations confer non-uniform effects on potency (NEP), i.e., larger effects for some volatile anesthetics than for others. Members of this class are of special interest for studies of arousal and its pharmacological suppression because they not only avoid the pitfall of effects on global health, but also imply the existence of drug targets that are preferentially affected by particular agents. In this work, we provide the first systematic investigation of the relative frequency and diversity of NEP mutations in Drosophila. As a first step, we isolated and characterized a set of P element insertion mutations that confer altered sensitivity of the fruit fly to the clinical anesthetic halothane. Then we tested the members of this collection for their effect on the sensitivity of flies to five other volatile agents. Not only do we find that most of the mutations show non-uniform effects, they also share a characteristic arrangement of altered potencies (halothane > >desflurane >or= enflurane approximately isoflurane approximately methoxyflurane > sevoflurane). From this result, although we do not know how direct or indirect are the effects of the mutations, we infer the existence of a biologically relevant target for anesthetic action that has a distinct preference for halothane over other agents. Intriguingly, P element insertions that co-map with several NEP loci have been shown to alter the fly's response to cocaine and ethanol, suggesting that common genetic elements are involved in the response to all three drugs.

Impact of gene copy number variation on anesthesia in Drosophila melanogaster.

BACKGROUND: Chromosomal deletions and duplications, which result in halving or doubling of copy number in a block of genes, are an important source of variation between individuals. Phenotypic effects of copy number variation are commonly observed, but effects on sensitivity to volatile anesthetics have not been assessed in any organism. METHODS: The potency with which halothane depresses the righting reflex of fruit flies was measured in congenic Drosophila strains, each of which was heterozygous for a deletion of average size 400 kb. Over 200 strains were examined, thereby scanning approximately half of the fly genome. RESULTS: Although the vast majority of deletion heterozygotes were indistinguishable from the control, eight had significantly altered sensitivity to halothane. Genetic tests supported the hypothesis that the change in anesthetic sensitivity was the result of reduction in copy number and not adventitious mutations in the strains. Among the eight outliers, the difference in halothane potency ranged from a 25% increase to a 15% decrease. Changes of similar magnitude but distinctive patterns were found when these lines were tested with enflurane, isoflurane, and sevoflurane. CONCLUSIONS: Variation in gene copy number has a significant impact on anesthetic sensitivity in Drosophila melanogaster. The level of transcription of a few genes must thus be limiting for a normal response to volatiles. Coupling between gene copy and gene expression is universal, and the components of the fly's nervous system are highly conserved; therefore, this work provides a rationale for investigating the clinical impact of copy number variation.

An unusual cation channel mediates photic control of locomotion in Drosophila.

A unique family of putative ion channels that are related to voltage-gated sodium and calcium channels has been identified in genomic and cDNA studies of metazoans. Aside from evidence for expression of family members in the nervous system, little is known about the operation of the channel or its functional significance. In the present study, this conserved family's sole Drosophila member, a gene known both as CG1517 and as Dmalpha1U, is shown to correspond to the narrow abdomen (na) gene and is the locus of a set of mutations that affect sensitivity to anesthetics. Immunohistochemistry of adult heads reveals that the channel is expressed in the neuropil of the central complex and optic lobe; expression is severely depressed in the mutants. In addition to previously described defects, the mutant phenotype is demonstrated here to include dysfunction in the coupling between light and locomotor behavior. Most dramatically, mutant flies have an inversion of relative locomotor activity in light versus dark. The involvement of the channel in daily rhythms of the fruit fly is especially provocative because the human ortholog lies in a candidate region linked to bipolar disorder, a disease frequently associated with altered diurnal behavior.

The mushroom body defect gene product is an essential component of the meiosis II spindle apparatus in Drosophila oocytes.

In addition to their well-known effects on the development of the mushroom body, mud mutants are also female sterile. Here we show that, although the early steps of ovary development are grossly normal, a defect becomes apparent in meiosis II when the two component spindles fail to cohere and align properly. The products of meiosis are consequently mispositioned within the egg and, with or without fertilization, soon undergo asynchronous and spatially disorganized replication. In wild-type eggs, Mud is found associated with the central spindle pole body that lies between the two spindles of meiosis II. The mutant defect thus implies that Mud should be added to the short list of components that are required for the formation and/or stability of this structure. Mud protein is also normally found in association with other structures during egg development: at the spindle poles of meiosis I, at the spindle poles of early cleavage and syncytial embryos, in the rosettes formed from the unfertilized products of meiosis, with the fusomes and spectrosomes that anchor the spindles of dividing cystoblasts, and at the nuclear rim of the developing oocyte. In contrast to its important role at the central spindle pole body, in none of these cases is it clear that Mud plays an essential role. But the commonalities in its location suggest potential roles for the protein in development of other tissues.

The role of TDP1 from budding yeast in the repair of DNA damage.

The TDP1 gene encodes a protein that can hydrolyze certain types of 3'-terminal phosphodiesters, but the relevance of these catalytic activities to gene function has not been previously tested. In this work we engineered a point mutation in TDP1 and present evidence that, as per design, it severely diminishes tyrosyl-DNA phosphodiesterase enzyme activity without affecting protein folding. The phenotypes of yeast strains that express this mutant show that the contribution of TDP1 to the repair of two kinds of damaged termini-induced, respectively, by camptothecin (CPT) and by bleomycin-strongly depends on enzyme activity. In routine assays of cell survival and growth the contribution of this activity is often overshadowed by other repair pathways. However, the value of TDP1 in the economy of the cell is highlighted by our discovery of several phenotypes that are evident even without deliberate inactivation of parallel pathways. These non-redundant mutant phenotypes include increased spontaneous mutation rate, transient accumulation of cells in a mid-anaphase checkpoint after exposure to camptothecin and, in cells that overexpress topoisomerase I (Top1), decreased survival of camptothecin-induced damage. The relationship between the role of TDP1 in Saccharomyces and its role in metazoans is discussed.

UNC79 and UNC80, putative auxiliary subunits of the NARROW ABDOMEN ion channel, are indispensable for robust circadian locomotor rhythms in Drosophila.

In the fruit fly Drosophila melanogaster, a network of circadian pacemaker neurons drives daily rhythms in rest and activity. The ion channel NARROW ABDOMEN (NA), orthologous to the mammalian sodium leak channel NALCN, functions downstream of the molecular circadian clock in pacemaker neurons to promote behavioral rhythmicity. To better understand the function and regulation of the NA channel, we have characterized two putative auxiliary channel subunits in Drosophila, unc79 (aka dunc79) and unc80 (aka CG18437). We have generated novel unc79 and unc80 mutations that represent strong or complete loss-of-function alleles. These mutants display severe defects in circadian locomotor rhythmicity that are indistinguishable from na mutant phenotypes. Tissue-specific RNA interference and rescue analyses indicate that UNC79 and UNC80 likely function within pacemaker neurons, with similar anatomical requirements to NA. We observe an interdependent, post-transcriptional regulatory relationship among the three gene products, as loss of na, unc79, or unc80 gene function leads to decreased expression of all three proteins, with minimal effect on transcript levels. Yet despite this relationship, we find that the requirement for unc79 and unc80 in circadian rhythmicity cannot be bypassed by increasing NA protein expression, nor can these putative auxiliary subunits substitute for each other. These data indicate functional requirements for UNC79 and UNC80 beyond promoting channel subunit expression. Immunoprecipitation experiments also confirm that UNC79 and UNC80 form a complex with NA in the Drosophila brain. Taken together, these data suggest that Drosophila NA, UNC79, and UNC80 function together in circadian clock neurons to promote rhythmic behavior.

Drosophila TRP channels require a protein with a distinctive motif encoded by the inaF locus.

In both vertebrates and invertebrates, ion channels of the TRP superfamily are known to be influenced by a variety of accessory factors, but the list of interacting proteins is acknowledged to be incomplete. Although previous work showed that Drosophila TRP function is disrupted by mutations in the inaF locus, the mechanism of this effect has remained obscure. Here we show that a previously overlooked small protein, INAF-B, is encoded by the locus and fulfills its critical role in retinal physiology. The 81-aa INAF-B gene product is an integral membrane protein that colocalizes to rhabdomeres along with TRP channels. Immunoprecipitation experiments demonstrate that the two proteins participate in a complex, and blotting experiments show that neither protein survives in the absence of the other. Both proteins are normally part of a large supramolecular assembly, the signalplex, but their interaction persists even in the absence of the scaffold for this structure. The inaF locus encodes three other proteins, each of which has diverged from INAF-B except for a 32-aa block of residues that encompasses a transmembrane domain. This conserved sequence defines an inaF motif, representatives of which are found in proteins from organisms as diverse as nematodes, fish, and humans. Given the role of INAF-B, these proteins are good candidates for interacting partners of other members of the TRP superfamily.

Retrograde signaling from the brain to the retina modulates the termination of the light response in Drosophila.

A critical factor in visual function is the speed with which photoreceptors (PRs) return to the resting state when light intensity dims. Several elements subserve this process, many of which promote the termination of the phototransduction cascade. Although the known elements are intrinsic to PRs, we have found that prompt restoration to the resting state of the Drosophila electroretinogram can require effective communication between the retina and the underlying brain. The requirement is seen more dramatically with long than with short light pulses, distinguishing the phenomenon from gross disruption of the termination machinery. The speed of recovery is affected by mutations (in the Hdc and ort genes) that prevent PRs from transmitting visual information to the brain. It is also affected by manipulation (using either drugs like neostigmine or genetic tools to inactivate neurotransmitter release) of cholinergic signals that arise in the brain. Intracellular recordings support the hypothesis that PRs are the target of this communication. We infer that signaling from the retina to the optic lobe prompts a feedback signal to retinal PRs. Although the mechanism of this retrograde signaling remains to be discerned, the phenomenon establishes a previously unappreciated mode of control of the temporal responsiveness of a primary sensory neuron.

A specific alteration in the electroretinogram of Drosophila melanogaster is induced by halothane and other volatile general anesthetics.

UNLABELLED: In higher organisms, physiological investigations have provided a valuable complement to assays of anesthetic effects on whole-animal behavior. However, although complex motor programs of Drosophila melanogaster have been used to identify genes that influence anesthesia, electrophysiological studies of anesthetic effects in this invertebrate have been limited. Here we show that the electroretinogram (ERG), the extracellular recording of light-evoked mass potentials from the surface of the eye, reveals a distinct effect of halothane, enflurane, isoflurane, and desflurane. Behaviorally relevant concentrations of these volatile anesthetics severely reduced the transient component of the ERG at lights-off. Other prominent ERG components, such as the photoreceptor potential and the lights-on transient, were not consistently affected by these drugs. Surprisingly, for most anesthetics, a diminished off-transient was obtained only with short light pulses. An identical effect was observed in the absence of anesthetic by depressing the function of Shaker potassium channels. The possibility that halothane acts in the visual circuit by closing potassium channels was examined with a simple genetic test; the results were consistent with the hypothesis but fell short of providing definitive support. Nevertheless, our studies establish the ERG as a useful tool both for examining the influence of volatile anesthetics on a simple circuit and for identifying genes that contribute to anesthetic sensitivity. IMPLICATIONS: Electroretinography (ERG) provides a useful monitor of anesthetic effects on the fruit fly. The effects of volatile anesthetics on the ERG are recapitulated by inactivation of potassium channels.

Repair of topoisomerase I covalent complexes in the absence of the tyrosyl-DNA phosphodiesterase Tdp1.

Accidental or drug-induced interruption of the breakage and reunion cycle of eukaryotic topoisomerase I (Top1) yields complexes in which the active site tyrosine of the enzyme is covalently linked to the 3' end of broken DNA. The enzyme tyrosyl-DNA phosphodiesterase (Tdp1) hydrolyzes this protein-DNA link and thus functions in the repair of covalent complexes, but genetic studies in yeast show that alternative pathways of repair exist. Here, we have evaluated candidate genes for enzymes that might act in parallel to Tdp1 so as to generate free ends of DNA. Despite finding that the yeast Apn1 protein has a Tdp1-like biochemical activity, genetic inactivation of all known yeast apurinic endonucleases does not increase the sensitivity of a tdp1 mutant to direct induction of Top1 damage. In contrast, assays of growth in the presence of the Top1 poison camptothecin (CPT) indicate that the structure-specific nucleases dependent on RAD1 and MUS81 can contribute independently of TDP1 to repair, presumably by cutting off a segment of DNA along with the topoisomerase. However, cells in which all three enzymes are genetically inactivated are not as sensitive to the lethal effects of CPT as are cells defective in double-strand break repair. We show that the MRE11 gene is even more critical than the RAD52 gene for double-strand break repair of CPT lesions, and comparison of an mre11 mutant with a tdp1 rad1 mus81 triple mutant demonstrates that other enzymes complementary to Tdp1 remain to be discovered.