Heterozygous Flies Research Articles

Heterozygous Variants in KCNJ10 Cause Paroxysmal Kinesigenic Dyskinesia Via Haploinsufficiency.

Most paroxysmal kinesigenic dyskinesia (PKD) cases are hereditary, yet approximately 60% of patients remain genetically undiagnosed. We undertook the present study to uncover the genetic basis for undiagnosed PKD patients. Whole-exome sequencing was performed for 106 PRRT2-negative PKD probands. The functional impact of the genetic variants was investigated in HEK293T cells and Drosophila. Heterozygous variants in KCNJ10 were identified in 11 individuals from 8 unrelated families, which accounted for 7.5% (8/106) of the PRRT2-negative probands. Both co-segregation of the identified variants and the significantly higher frequency of rare KCNJ10 variants in PKD cases supported impacts from the detected KCNJ10 heterozygous variants on PKD pathogenesis. Moreover, a KCNJ10 mutation-carrying father from a typical EAST/SeSAME family was identified as a PKD patient. All patients manifested dystonia attacks triggered by sudden movement with a short episodic duration. Patch-clamp recordings in HEK293T cells revealed apparent reductions in K+ currents of the patient-derived variants, indicating a loss-of-function. In Drosophila, milder hyperexcitability phenotypes were observed in heterozygous Irk2 knock-in flies compared to homozygotes, supporting haploinsufficiency as the mechanism for the detected heterozygous variants. Electrophysiological recordings showed that excitatory neurons in Irk2 haploinsufficiency flies exhibited increased excitability, and glia-specific complementation with human Kir4.1 rescued the Irk2 mutant phenotypes. Our study established haploinsufficiency resulting from heterozygous variants in KCNJ10 can be understood as a previously unrecognized genetic cause for PKD and provided evidence of glial involvement in the pathophysiology of PKD. ANN NEUROL 2024;96:758-773.

Knock-in mutagenesis in Drosophila Rdl underscores the critical role of the conserved M3 glycine in mediating the actions of broflanilide and isocycloseram on GABA receptors

γ-Aminobutyric acid receptors (GABARs) are crucial targets for pest control chemicals, including meta-diamide and isoxazoline insecticides, which act as negative allosteric modulators of insect GABARs. Previous cell-based assays have indicated that amino acid residues in the transmembrane cavity between adjacent subunits of Drosophila RDL GABAR (i.e., Ile276, Leu280, and Gly335) are involved in mediating the action of meta-diamides. In this study, to confirm this result at the organismal level, we employed CRISPR/Cas9-mediated genome editing, generated six transgenic Drosophila strains carrying substitutions in these amino acid residues, and investigated their sensitivity to broflanilide and isocycloseram. Flies homozygous for the I276F mutation did not exhibit any change in sensitivity to the tested insecticides compared to the control flies. Conversely, I276C homozygosity was lethal, and heterozygous flies exhibited ∼2-fold lower sensitivity to broflanilide than the control flies. Flies homozygous for the L280C mutation survived into adulthood but exhibited infertility. Both heterozygous and homozygous L280C flies exhibited ∼3- and ∼20-fold lower sensitivities to broflanilide and isocycloseram, respectively, than the control flies. The reduction in sensitivity to isocycloseram in L280C flies diminished to ∼3-fold when treated with piperonyl butoxide. Flies homozygous for the G335A mutation reached the adult stage. However, they were sterile, had small bodies, and exhibited reduced locomotion, indicating the critical role of Gly335 in RDL function. These flies exhibited markedly increased tolerance to topically applied broflanilide and isocycloseram, demonstrating that the conserved Gly335 is the target of the insecticidal actions of broflanilide and isocycloseram. Considering the significant fitness costs, the Gly335 mutation may not pose a serious risk for the development of resistance in field populations of insect pests. However, more careful studies using insect pests are needed to investigate whether our perspective applies to resistance development under field conditions.

SREBP modulates the NADP+/NADPH cycle to control night sleep in Drosophila

Sleep behavior is conserved throughout evolution, and sleep disturbances are a frequent comorbidity of neuropsychiatric disorders. However, the molecular basis underlying sleep dysfunctions in neurological diseases remains elusive. Using a model for neurodevelopmental disorders (NDDs), the Drosophila Cytoplasmic FMR1 interacting protein haploinsufficiency (Cyfip85.1/+), we identify a mechanism modulating sleep homeostasis. We show that increased activity of the sterol regulatory element-binding protein (SREBP) in Cyfip85.1/+ flies induces an increase in the transcription of wakefulness-associated genes, such as the malic enzyme (Men), causing a disturbance in the daily NADP+/NADPH ratio oscillations and reducing sleep pressure at the night-time onset. Reduction in SREBP or Men activity in Cyfip85.1/+ flies enhances the NADP+/NADPH ratio and rescues the sleep deficits, indicating that SREBP and Men are causative for the sleep deficits in Cyfip heterozygous flies. This work suggests modulation of the SREBP metabolic axis as a new avenue worth exploring for its therapeutic potential in sleep disorders.

Caffeine Delays Ethanol-Induced Sedation in Drosophila.

Caffeine and ethanol are among the most widely available and commonly consumed psychoactive substances. Both interact with adenosine receptor-mediated signaling which regulates numerous neurological processes including sleep and waking behaviors. In mammals, caffeine is an adenosine receptor antagonist and thus acts as a stimulant. Conversely, ethanol is a sedative because it promotes GABAergic neurotransmission, inhibits glutamatergic neurotransmission, and increases the amount of adenosine in the brain. Despite seemingly overlapping interactions, not much is known about the effect of caffeine on ethanol-induced sedation in Drosophila. In this study, using Drosophila melanogaster as a model, we show that caffeine supplementation in food delays the onset of ethanol-induced sedation in males and females of different strains. The resistance to sedation reverses upon caffeine withdrawal. Heterozygous adenosine receptor mutant flies are resistant to sedation. These findings suggest that caffeine and adenosine receptors modulate the sedative effects of ethanol in Drosophila.

Evolution of casein kinase 1 and functional analysis of new doubletime mutants in Drosophila.

Circadian clocks are timing devices that rhythmically adjust organism's behavior, physiology, and metabolism to the 24-h day-night cycle. Eukaryotic circadian clocks rely on several interlocked transcription-translation feedback loops, where protein stability is the key part of the delay between transcription and the appearance of the mature proteins within the feedback loops. In bilaterian animals, including mammals and insects, the circadian clock depends on a homologous set of proteins. Despite mostly conserved clock components among the fruit fly Drosophila and mammals, several lineage-specific differences exist. Here we have systematically explored the evolution and sequence variability of insect DBT proteins and their vertebrate homologs casein kinase 1 delta (CKIδ) and epsilon (CKIε), dated the origin and separation of CKIδ from CKIε, and identified at least three additional independent duplications of the CKIδ/ε gene in Petromyzon, Danio, and Xenopus. We determined conserved regions in DBT specific to Diptera, and functionally tested a subset of those in D. melanogaster. Replacement of Lysine K224 with acidic residues strongly impacts the free-running period even in heterozygous flies, whereas homozygous mutants are not viable. K224D mutants have a temperature compensation defect with longer free-running periods at higher temperatures, which is exactly the opposite trend of what was reported for corresponding mammalian mutants. All DBTs of dipteran insects contain the NKRQK motif at positions 220-224. The occurrence of this motif perfectly correlates with the presence of BRIDE OF DOUBLETIME, BDBT, in Diptera. BDBT is a non-canonical FK506-binding protein that physically interacts with Drosophila DBT. The phylogeny of FK506-binding proteins suggests that BDBT is either absent or highly modified in non-dipteran insects. In addition to in silico analysis of DBT/CKIδ/ε evolution and diversity, we have identified four novel casein kinase 1 genes specific to the Drosophila genus.

A Drosophila Su(H) model of Adams-Oliver Syndrome reveals cofactor titration as a mechanism underlying developmental defects

Notch signaling is a conserved pathway that converts extracellular receptor-ligand interactions into changes in gene expression via a single transcription factor (CBF1/RBPJ in mammals; Su(H) in Drosophila). In humans, RBPJ variants have been linked to Adams-Oliver syndrome (AOS), a rare autosomal dominant disorder characterized by scalp, cranium, and limb defects. Here, we found that a previously described Drosophila Su(H) allele encodes a missense mutation that alters an analogous residue found in an AOS-associated RBPJ variant. Importantly, genetic studies support a model that heterozygous Drosophila with the AOS-like Su(H) allele behave in an opposing manner to heterozygous flies with a Su(H) null allele, due to a dominant activity of sequestering either the Notch co-activator or the antagonistic Hairless co-repressor. Consistent with this model, AOS-like Su(H) and Rbpj variants have decreased DNA binding activity compared to wild type proteins, but these variants do not significantly alter protein binding to the Notch co-activator or the fly and mammalian co-repressors, respectively. Taken together, these data suggest a cofactor sequestration mechanism underlies AOS phenotypes associated with RBPJ variants, whereby the AOS-associated RBPJ allele encodes a protein with compromised DNA binding activity that retains cofactor binding, resulting in Notch target gene dysregulation.

Dual Oxidase, a Hydrogen-Peroxide-Producing Enzyme, Regulates Neuronal Oxidative Damage and Animal Lifespan in Drosophila melanogaster.

Reducing the oxidative stress in neurons extends lifespan in Drosophila melanogaster, highlighting the crucial role of neuronal oxidative damage in lifespan determination. However, the source of the reactive oxygen species (ROS) that provoke oxidative stress in neurons is not clearly defined. Here, we identify dual oxidase (duox), a calcium-activated ROS-producing enzyme, as a lifespan determinant. Due to the lethality of duox homozygous mutants, we employed a duox heterozygote that exhibited normal appearance and movement. We found that duox heterozygous male flies, which were isogenized with control flies, demonstrated extended lifespan. Neuronal knockdown experiments further suggested that duox is crucial to oxidative stress in neurons. Our findings suggest duox to be a source of neuronal oxidative stress associated with animal lifespan.

FTD-associated mutations in Tau result in a combination of dominant and recessive phenotypes

Although mutations in the microtubules-associated protein Tau have long been connected with several neurodegenerative diseases, the underlying molecular mechanisms causing these tauopathies are still not fully understood. Studies in various models suggested that dominant gain-of-function effects underlie the pathogenicity of these mutants; however, there is also evidence that the loss of normal physiological functions of Tau plays a role in tauopathies. Previous studies on Tau in Drosophila involved expressing the human Tau protein in the background of the endogenous Tau gene in addition to inducing high expression levels. To study Tau pathology in more physiological conditions, we recently created Drosophila knock-in models that express either wildtype human Tau (hTauWT) or disease-associated mutant hTau (hTauV337M and hTauK369I) in place of the endogenous Drosophila Tau (dTau). Analyzing these flies as homozygotes, we could therefore detect recessive effects of the mutations while identifying dominant effects in heterozygotes. Using memory, locomotion and sleep assays, we found that homozygous mutant hTau flies showed deficits already when quite young whereas in heterozygous flies, disease phenotypes developed with aging. Homozygotes also revealed an increase in microtubule diameter, suggesting that changes in the cytoskeleton underlie the axonal degeneration we observed in these flies. In contrast, heterozygous mutant hTau flies showed abnormal axonal targeting and no detectable changes in microtubules. However, we previously showed that heterozygosity for hTauV337M interfered with synaptic homeostasis in central pacemaker neurons and we now show that heterozygous hTauK369I flies have decreased levels of proteins involved in the release of synaptic vesicles. Taken together, our results demonstrate that both mutations induce a combination of dominant and recessive disease-related phenotypes that provide behavioral and molecular insights into the etiology of Tauopathies.

Drosophila carrying epilepsy-associated variants in the vitamin B6 metabolism gene PNPO display allele- and diet-dependent phenotypes

Pyridox(am)ine 5 ' -phosphate oxidase (PNPO) catalyzes the rate-limiting step in the synthesis of pyridoxal 5 ' -phosphate (PLP), the active form of vitamin B6 required for the synthesis of neurotransmitters gamma-aminobutyric acid (GABA) and the monoamines. Pathogenic variants in PNPO have been increasingly identified in patients with neonatal epileptic encephalopathy and early-onset epilepsy. These patients often exhibit different types of seizures and variable comorbidities. Recently, the PNPO gene has also been implicated in epilepsy in adults. It is unclear how these phenotypic variations are linked to specific PNPO alleles and to what degree diet can modify their expression. Using CRISPR-Cas9, we generated four knock-in Drosophila alleles, hWT , hR116Q , hD33V , and hR95H , in which the endogenous Drosophila PNPO was replaced by wild-type human PNPO complementary DNA (cDNA) and three epilepsy-associated variants. We found that these knock-in flies exhibited a wide range of phenotypes, including developmental impairments, abnormal locomotor activities, spontaneous seizures, and shortened life span. These phenotypes are allele dependent, varying with the known biochemical severity of these mutations and our characterized molecular defects. We also showed that diet treatments further diversified the phenotypes among alleles, and PLP supplementation at larval and adult stages prevented developmental impairments and seizures in adult flies, respectively. Furthermore, we found that hR95H had a significant dominant-negative effect, rendering heterozygous flies susceptible to seizures and premature death. Together, these results provide biological bases for the various phenotypes resulting from multifunction of PNPO, specific molecular and/or genetic properties of each PNPO variant, and differential allele-diet interactions.

The short neuropeptide F receptor regulates olfaction-mediated foraging behavior in the oriental fruit fly Bactrocera dorsalis (Hendel)

The short neuropeptide F (sNPF) signaling system, consisting of sNPF and its receptor (sNPFR), influences many physiological processes in insects, including feeding, growth and olfactory memory. We previously showed that sNPF regulates olfactory sensitivity in the oriental fruit fly Bactrocera dorsalis (Hendel) during starvation. However, the functional analysis of sNPFR is constrained by the failure of RNA interference in this species. Here, we generated a null sNPFR mutant using the CRISPR/Cas9 system to investigate the physiological roles of this receptor in more detail. G0 adults were produced at a frequency of 60.8%, and sNPFR−/− mutants were obtained after several generations of backcrossing followed by self-crossing among heterozygous flies. We found that the mutants were significantly less successful at foraging for certain foods and showed increased foraging latency. Electroantennogram (EAG) assays indicated that the mutants had significantly lower electrophysiological responses to three tested odorants. Furthermore, qPCR data revealed the inhibition of several olfactory receptor genes, including Orco. Immunohistochemistry showed that BdsNPFR was localized in cells under the sensillum on the antennae. Based on their shape and size, the BdsNPFR+ cells differ from odorant receptor neurons (ORNs), which were labeled using a Drosophila melanogaster Orco antibody. Our data suggest that sNPFR regulates olfaction-mediated foraging behavior by mediating interactions between BdsNPFR+ cells and selected ORNs.

Biological effects of low-dose γ-ray irradiation on chromosomes and DNA of Drosophila melanogaster.

While the damage to chromosomes and genes induced by high-dose radiation (HDR) has been well researched in many organisms, the effects of low-dose radiation (LDR), defined as a radiation dose of ≤100 mSv, are still being debated. Recent research has suggested that the biological effects of LDR differ from those observed in HDR. To detect the effect of LDR on genes, we selected a gene of Drosophila melanogaster, known as the multiple wing hair (mwh) gene. The hatched heterozygous larvae with genotype mwh/+ were irradiated by γ-rays of a 60Co source. After eclosion, the wing hairs of the heterozygous flies were observed. The area of only one or two mwh cells (small spot) and that of more than three mwh cells (large spot) were counted. The ratio of the two kinds of spots were compared between groups irradiated by different doses including a non-irradiated control group. For the small spot in females, the eruption frequency increased in the groups irradiated with 20–75 mGy, indicating hypersensitivity (HRS) to LDR, while in the groups irradiated with 200 and 300 mGy, the frequency decreased, indicating induced radioresistance (IRR), while in males, 50 and 100 mGy conferred HRS and 75 and 200 mGy conferred IRR. For the large spot in females, 75 mGy conferred HRS and 100–800 mGy conferred IRR. In conclusion, HRS and IRR to LDR was found in Drosophila wing cells by delimiting the dose of γ-rays finely, except in the male large spot.

Mitochondrial Complex I Mutations Predispose Drosophila to Isoflurane Neurotoxicity.

General anesthetics influence mitochondrial homeostasis, placing individuals with mitochondrial disorders and possibly carriers of recessive mitochondrial mutations at increased risk of perioperative complications. In Drosophila, mutations in the ND23 subunit of complex I of the mitochondrial electron transport chain-analogous to mammalian NDUFS8-replicate key characteristics of Leigh syndrome, an inherited mitochondrial disorder. The authors used the ND23 mutant for testing the hypothesis that anesthetics have toxic potential in carriers of mitochondrial mutations. The authors exposed wild-type flies and ND23 mutant flies to behaviorally equivalent doses of isoflurane or sevoflurane in 5%, 21%, or 75% oxygen. The authors used percent mortality (mean ± SD, n ≥ 3) at 24 h after exposure as a readout of toxicity and changes in gene expression to investigate toxicity mechanisms. Exposure of 10- to 13-day-old male ND23 flies to isoflurane in 5%, 21%, or 75% oxygen resulted in 16.0 ± 14.9% (n = 10), 48.2 ± 16.1% (n = 9), and 99.2 ± 2.0% (n = 10) mortality, respectively. Comparable mortality was observed in females. In contrast, under the same conditions, mortality was less than 5% for all male and female groups exposed to sevoflurane, except 10- to 13-day-old male ND23 flies with 9.6 ± 8.9% (n = 16) mortality. The mortality of 10- to 13-day-old ND23 flies exposed to isoflurane was rescued by neuron- or glia-specific expression of wild-type ND23. Isoflurane and sevoflurane differentially affected expression of antioxidant genes in 10- to 13-day-old ND23 flies. ND23 flies had elevated mortality from paraquat-induced oxidative stress compared with wild-type flies. The mortality of heterozygous ND23 flies exposed to isoflurane in 75% oxygen increased with age, resulting in 54.0 ± 19.6% (n = 4) mortality at 33 to 39 days old, and the percent mortality varied in different genetic backgrounds. Mutations in the mitochondrial complex I subunit ND23 increase susceptibility to isoflurane-induced toxicity and to oxidative stress in Drosophila. Asymptomatic flies that carry ND23 mutations are sensitized to hyperoxic isoflurane toxicity by age and genetic background.

Drosophila Uev1a is dually required for Ben-dependent DNA-damage response and fly mobility

K63-linked polyubiquitination requires the ubiquitin-conjugating enzyme Ubc13 and a Ubc/E2 variant Uev. Lower eukaryotic organisms contain one UEV gene required for DNA-damage tolerance, while vertebrates and higher plants contain multiple UEV genes with distinct functions. In contrast, Drosophila contains only one UEV gene designated dUev1a. Here we report that dUev1a forms a stable heterodimer with Ben, the Drosophila Ubc13 ortholog, that dUev1a-F15E completely abolishes the interaction, and that a conserved dUev1a-F15Y substitution severely reduces its interaction with Ben. dUev1a functionally rescues the corresponding yeast mms2 null mutant from killing by various DNA-damaging agents in a Ben-dependent manner, and the heterozygous dUev1a mutant flies are more sensitive to DNA-damaging agent, indicating that the function of UEV in DNA-damage response is conserved throughout eukaryotes. Meanwhile, dUev1a+/‐ mutant flies displayed reduced mobility characteristic of defects in the central nervous system and reminiscent of the bendless phenotypes, suggesting that dUev1a acts together with Ben in this process. Our observations collectively imply that dUev1a is dually required for DNA-damage response and neurological signaling in Drosophila, and that these processes are mediated by the Ben-dUev1a complex that promotes K63-linked polyubiquitination.

Disease-Associated Mutant Tau Prevents Circadian Changes in the Cytoskeleton of Central Pacemaker Neurons.

A hallmark feature of Alzheimer’s disease (AD) and other Tauopathies, like Frontotemporal Dementia with Parkinsonism linked to chromosome 17 (FTDP-17), is the accumulation of neurofibrillary tangles composed of the microtubule-associated protein Tau. As in AD, symptoms of FTDP-17 include cognitive decline, neuronal degeneration, and disruptions of sleep patterns. However, mechanisms by which Tau may lead to these disturbances in sleep and activity patterns are unknown. To identify such mechanisms, we have generated novel Drosophila Tauopathy models by replacing endogenous fly dTau with normal human Tau (hTau) or the FTDP-17 causing hTauV337M mutation. This mutation is localized in one of the microtubule-binding domains of hTau and has a dominant effect. Analyzing heterozygous flies, we found that aged hTauV337M flies show neuronal degeneration and locomotion deficits when compared to wild type or hTauWT flies. Furthermore, hTauV337M flies are hyperactive and they show a fragmented sleep pattern. These changes in the sleep/activity pattern are accompanied by morphological changes in the projection pattern of the central pacemaker neurons. These neurons show daily fluctuations in their connectivity, whereby synapses are increased during the day and reduced during sleep. Synapse formation requires cytoskeletal changes that can be detected by the accumulation of the end-binding protein 1 (EB1) at the site of synapse formation. Whereas, hTauWT flies show the normal day/night changes in EB1 accumulation, hTauV337M flies do not show this fluctuation. This suggests that hTauV337M disrupts sleep patterns by interfering with the cytoskeletal changes that are required for the synaptic homeostasis of central pacemaker neurons.

The glutamic acid-rich–long C-terminal extension of troponin T has a critical role in insect muscle functions

The troponin complex regulates the Ca2+ activation of myofilaments during striated muscle contraction and relaxation. Troponin genes emerged 500-700 million years ago during early animal evolution. Troponin T (TnT) is the thin-filament-anchoring subunit of troponin. Vertebrate and invertebrate TnTs have conserved core structures, reflecting conserved functions in regulating muscle contraction, and they also contain significantly diverged structures, reflecting muscle type- and species-specific adaptations. TnT in insects contains a highly-diverged structure consisting of a long glutamic acid-rich C-terminal extension of ∼70 residues with unknown function. We found here that C-terminally truncated Drosophila TnT (TpnT-CD70) retains binding of tropomyosin, troponin I, and troponin C, indicating a preserved core structure of TnT. However, the mutant TpnTCD70 gene residing on the X chromosome resulted in lethality in male flies. We demonstrate that this X-linked mutation produces dominant-negative phenotypes, including decreased flying and climbing abilities, in heterozygous female flies. Immunoblot quantification with a TpnT-specific mAb indicated expression of TpnT-CD70 in vivo and normal stoichiometry of total TnT in myofilaments of heterozygous female flies. Light and EM examinations revealed primarily normal sarcomere structures in female heterozygous animals, whereas Z-band streaming could be observed in the jump muscle of these flies. Although TpnT-CD70-expressing flies exhibited lower resistance to cardiac stress, their hearts were significantly more tolerant to Ca2+ overloading induced by high-frequency electrical pacing. Our findings suggest that the Glu-rich long C-terminal extension of insect TnT functions as a myofilament Ca2+ buffer/reservoir and is potentially critical to the high-frequency asynchronous contraction of flight muscles.

The regulation of triglyceride storage by ornithine decarboxylase (Odc1) in Drosophila

Polyamines are low molecular weight, organic cations that play a critical role in many major cellular processes including cell cycle regulation and apoptosis, cellular division, tissue proliferation, and cellular differentiation; however, the functions of polyamines in regulating the storage of metabolic fuels such as triglycerides and glycogen is poorly understood. To address this question, we focused on the Drosophila homolog of ornithine decarboxylase (Odc1), the first rate-limiting enzyme in the synthesis of polyamines. Mutants in Odc1 are lethal, but heterozygotes were viable to adulthood. Odc1 heterozygotes appeared larger than their genetic background control flies and consistent with this observation, weighed more than the controls. However, the increased weight was not due to increased food consumption as heterozygotes ate less than the controls. Interestingly, Odc1 heterozygous flies had augmented triglyceride storage, and this lipid phenotype was due to increased triglyceride storage per cell and an increase in the number of fat cells produced. Odc1 heterozygous flies also displayed increased expression of the lipid synthesis genes fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC), suggesting increased lipid synthesis was the cause of the augmented triglyceride phenotype. These results provide a link between the expression of Odc1 and triglyceride storage suggesting that the polyamine pathway plays a role in regulating lipid metabolism.

Genetic interactions between Protein Kinase D and Lobe mutants during eye development of Drosophila melanogaster

BackgroundIn Drosophila, the development of the fly eye involves the activity of several, interconnected pathways that first define the presumptive eye field within the eye anlagen, followed by establishment of the dorso-ventral boundary, and the regulation of growth and apoptosis. In Lobe (L) mutant flies, parts of the eye or even the complete eye are absent because the eye field has not been properly defined. Manifold genetic interactions indicate that L influences the activity of several signalling pathways, resulting in a conversion of eye tissue into epidermis, and in the induction of apoptosis. As information on the molecular nature of the L mutation is lacking, the underlying molecular mechanisms are still an enigma.ResultsWe have identified Protein Kinase D (PKD) as a strong modifier of the L mutant phenotype. PKD belongs to the PKC/CAMK class of Ser/Thr kinases that have been involved in diverse cellular processes including stress resistance and growth. Despite the many roles of PKD, Drosophila PKD null mutants are without apparent phenotype apart from sensitivity to oxidative stress. Here we report an involvement of PKD in eye development in the sensitized genetic background of Lobe. Absence of PKD strongly enhanced the dominant eye defects of heterozygous L2 flies, and decreased their viability. Moreover, eye-specific overexpression of an activated isoform of PKD considerably ameliorated the dominant L2 phenotype. This genetic interaction was not allele specific but similarly seen with three additional, weaker L alleles (L1, L5, LG), demonstrating its specificity.ConclusionsWe propose that PKD-mediated phosphorylation is involved in underlying processes causing the L phenotype, i.e. in the regulation of growth, the epidermal transformation of eye tissue and apoptosis, respectively.

The R249Q hypertrophic cardiomyopathy myosin mutation decreases contractility in Drosophila by impeding force production.

Hypertrophic cardiomyopathy (HCM) is a genetic disease that causes thickening of the heart's ventricular walls and is a leading cause of sudden cardiac death. HCM is caused by missense mutations in muscle proteins including myosin, but how these mutations alter muscle mechanical performance in largely unknown. We investigated the disease mechanism for HCM myosin mutation R249Q by expressing it in the indirect flight muscle of Drosophila melanogaster and measuring alterations to muscle and flight performance. Muscle mechanical analysis revealed R249Q decreased muscle power production due to slower muscle kinetics and decreased force production; force production was reduced because fewer mutant myosin cross-bridges were bound simultaneously to actin. This work does not support the commonly proposed hypothesis that myosin HCM mutations increase muscle contractility, or causes a gain in function; instead, it suggests that for some myosin HCM mutations, hypertrophy is a compensation for decreased contractility. Hypertrophic cardiomyopathy (HCM) is an inherited disease that causes thickening of the heart's ventricular walls. A generally accepted hypothesis for this phenotype is that myosin heavy chain HCM mutations increase muscle contractility. To test this hypothesis, we expressed an HCM myosin mutation, R249Q, in Drosophila indirect flight muscle (IFM) and assessed myofibril structure, skinned fibre mechanical properties, and flight ability. Mechanics experiments were performed on fibres dissected from 2-h-old adult flies, prior to degradation of IFM myofilament structure, which started at 2days old and increased with age. Homozygous and heterozygous R249Q fibres showed decreased maximum power generation by 67% and 44%, respectively. Decreases in force and work and slower overall muscle kinetics caused homozygous fibres to produce less power. While heterozygous fibres showed no overall slowing of muscle kinetics, active force and work production dropped by 68% and 47%, respectively, which hindered power production. The muscle apparent rate constant 2πb decreased 33% for homozygous but increased for heterozygous fibres. The apparent rate constant 2πc was greater for homozygous fibres. This indicates that R249Q myosin is slowing attachment while speeding up detachment from actin, resulting in less time bound. Decreased IFM power output caused 43% and 33% decreases in Drosophila flight ability and 19% and 6% drops in wing beat frequency for homozygous and heterozygous flies, respectively. Overall, our results do not support the increased contractility hypothesis. Instead, our results suggest the ventricular hypertrophy for human R249Q mutation is a compensatory response to decreases in heart muscle power output.

In Vivo Efficacy of Ellagic Acid against Candida albicans in a Drosophila melanogaster Infection Model.

The aim of this study was to evaluate the antifungal activity and the toxicity of ellagic acid (EA) using a Drosophila melanogaster model. Candida albicans bacteria were inoculated into Toll heterozygous flies. Survival curves were obtained for the evaluation of the antimicrobial effect and toxicity of EA. A protective effect of EA against fungal infection in Drosophila melanogaster was observed at nontoxic concentrations. This study showed that EA is a promising tool for the treatment of candidiasis.

Bisphenol A induced oxidative stress mediated genotoxicity in Drosophila melanogaster.

This study investigates Bisphenol A (BPA) induced oxidative stress that mediates the genotoxicity in in vivo model Drosophila melanogaster. The calculated LC50 for BPA was 12.35 μg/mL. The strains of D. melanogaster were reared in 0.1, 1.0, 2.5 and 5.0 μg/mL BPA treated food media from the embryonic stage (egg); oxidative stress and genotoxicity parameters were analyzed. Food intake analysis confirmed that BPA is not an anti feedant for Drosophila larvae and it consumed BPA containing food. Increased reactive oxygen species (ROS) and lipid peroxidation (LPO) and depletion of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and glutathione-s-transferase (GST) antioxidant activities were observed in BPA treated groups compared to control. Positive single spots/wing frequencies were observed in standard (ST) and high bioactivation (HB) crosses of marker heterozygous (MH; mwh/flr3) and balancer heterozygous (BH; mwh/TM3) genotype flies indicating BPA is mutagenic and not recombinogenic. A significant increase in tail length and % tail DNA in Comet assay after BPA treatment reveals that BPA has a potential to induce the genotoxicity. Present study suggests that BPA exposure induces oxidative stress, which could be one of the possible mechanisms for induction of genotoxicity.