In vivo expression of peptidylarginine deiminase in Drosophila melanogaster.

Peptidylarginine deiminase (PAD) modifies peptidylarginine and converts it to peptidylcitrulline in the presence of elevated calcium. Protein modification can lead to severe changes in protein structure and function, and aberrant PAD activity is linked to human pathologies. While PAD homologs have been discovered in vertebrates-as well as in protozoa, fungi, and bacteria-none have been identified in Drosophila melanogaster, a simple and widely used animal model for human diseases. Here, we describe the development of a human PAD overexpression model in Drosophila. We established fly lines harboring human PAD2 or PAD4 transgenes for ectopic expression under control of the GAL4/UAS system. We show that ubiquitous or nervous system expression of PAD2 or PAD4 have minimal impact on fly lifespan, fecundity, and the response to acute heat stress. Although we did not detect citrullinated proteins in fly homogenates, fly-expressed PAD4-but not PAD2-was active in vitro upon Ca2+ supplementation. The transgenic fly lines may be valuable in future efforts to develop animal models of PAD-related disorders and for investigating the biochemistry and regulation of PAD function.

Contribution of a natural polymorphism in protein kinase G modulates electroconvulsive seizure recovery in Drosophila melanogaster.

Drosophila melanogaster is a well-characterized model for neurological disorders and is widely used for investigating causes of altered neuronal excitability leading to seizure-like behavior. One method used to analyze behavioral output of neuronal perturbance is recording the time to locomotor recovery from an electroconvulsive shock. Based on this behavior, we sought to quantify seizure susceptibility in larval D. melanogaster with differences in the enzymatic activity levels of a major protein, cGMP-dependent protein kinase (PKG). PKG, encoded by foraging, has two natural allelic variants and has previously been implicated in several important physiological characteristics including: foraging patterns, learning and memory, and environmental stress tolerance. The well-established NO/cGMP/PKG signaling pathway found in the fly, which potentially targets downstream K+ channel(s), ultimately impacts membrane excitability, leading to our hypothesis: altering PKG enzymatic activity modulates time to recovery from an electroconvulsive seizure. Our results show that by both genetically and pharmacologically increasing PKG enzymatic activity, we can decrease the locomotor recovery time from an electroconvulsive seizure in larval D. melanogaster.

egl-4 modulates electroconvulsive seizure duration in C. elegans.

Increased neuronal excitability causes seizures with debilitating symptoms. Effective and noninvasive treatments are limited for easing symptoms, partially due to the complexity of the disorder and lack of knowledge of specific molecular faults. An unexplored, novel target for seizure therapeutics is the cGMP/protein kinase G (PKG) pathway, which targets downstream K+ channels, a mechanism similar to Retigabine, a recently FDA-approved antiepileptic drug. Our results demonstrate that increased PKG activity decreased seizure duration in C. elegans utilizing a recently developed electroconvulsive seizure assay. While the fly is a well-established seizure model, C. elegans are an ideal yet unexploited model which easily uptakes drugs and can be utilized for high-throughput screens. In this study, we show that treating the worms with either a potassium channel opener, Retigabine or published pharmaceuticals that increase PKG activity, significantly reduces seizure recovery times. Our results suggest that PKG signaling modulates downstream K+ channel conductance to control seizure recovery time in C. elegans. Hence, we provide powerful evidence, suggesting that pharmacological manipulation of the PKG signaling cascade may control seizure duration across phyla.

The HECT Family Ubiquitin Ligase EEL-1 Regulates Neuronal Function and Development.

Genetic changes in the HECT ubiquitin ligase HUWE1 are associated with intellectual disability, but it remains unknown whether HUWE1 functions in post-mitotic neurons to affect circuit function. Using genetics, pharmacology, and electrophysiology, we show that EEL-1, the HUWE1 ortholog in C. elegans, preferentially regulates GABAergic presynaptic transmission. Decreasing or increasing EEL-1 function alters GABAergic transmission and the excitatory/inhibitory (E/I) balance in the worm motor circuit, which leads to impaired locomotion and increased sensitivity to electroshock. Furthermore, multiple mutations associated with intellectual disability impair EEL-1 function. Although synaptic transmission defects did not result from abnormal synapse formation, sensitizing genetic backgrounds revealed that EEL-1 functions in the same pathway as the RING family ubiquitin ligase RPM-1 to regulate synapse formation and axon termination. These findings from a simple model circuit provide insight into the molecular mechanisms required to obtain E/I balance and could have implications for the link between HUWE1 and intellectual disability.

Electroshock Induced Seizures in Adult C. elegans.

The nematode Caenorhabditis elegans is a useful model organism for dissecting molecular mechanisms of neurological diseases. While hermaphrodite C. elegans contains only 302 neurons, the conserved homologous neurotransmitters, simpler neuronal circuitry, and fully mapped connectome make it an appealing model system for neurological research. Here we developed an assay to induce an electroconvulsive seizure in C. elegans which can be used as a behavioral method of analyzing potential anti-epileptic therapeutics and novel genes involved in seizure susceptibility. In this assay, worms are suspended in an aqueous solution as current is passed through the liquid. At the onset of the shock, worms will briefly paralyze and twitch, and shortly after regain normal sinusoidal locomotion. The time to locomotor recovery is used as a metric of recovery from a seizure which can be reduced or extended by incorporating drugs that alter neuronal and muscular excitability.

Modulating Behavior in C. elegans Using Electroshock and Antiepileptic Drugs.

The microscopic nematode Caenorhabditis elegans has emerged as a valuable model for understanding the molecular and cellular basis of neurological disorders. The worm offers important physiological similarities to mammalian models such as conserved neuron morphology, ion channels, and neurotransmitters. While a wide-array of behavioral assays are available in C. elegans, an assay for electroshock/electroconvulsion remains absent. Here, we have developed a quantitative behavioral method to assess the locomotor response following electric shock in C. elegans. Electric shock impairs normal locomotion, and induces paralysis and muscle twitching; after a brief recovery period, shocked animals resume normal locomotion. We tested electric shock responses in loss-of-function mutants for unc-25, which encodes the GABA biosynthetic enzyme GAD, and unc-49, which encodes the GABAA receptor. unc-25 and unc-49 mutants have decreased inhibitory GABAergic transmission to muscles, and take significantly more time to recover normal locomotion following electric shock compared to wild-type. Importantly, increased sensitivity of unc-25 and unc-49 mutants to electric shock is rescued by treatment with antiepileptic drugs, such as retigabine. Additionally, we show that pentylenetetrazol (PTZ), a GABAA receptor antagonist and proconvulsant in mammalian and C. elegans seizure models, increases susceptibility of worms to electric shock.

Pushing the limit: examining factors that affect anoxia tolerance in a single genotype of adult D. melanogaster.

Drosophila melanogaster is a promiscuous species that inhabits a large range of harsh environments including flooded habitats and varying temperature changes. To survive these environments, fruit flies have adapted mechanisms of tolerance that allow them to thrive. During exposure to anoxic stress, fruit flies and other poikilotherms enter into a reversible, protective coma. This coma can be manipulated based on controlled environmental conditions inside the laboratory. Here we utilize a common laboratory raised strain of D. melanogaster to characterize adaptation abilities to better understand coma recovery and survival limitations. Our goal is to mimic the fly's natural environments (wet anoxia) and relate findings to a typical gas induced environment (dry anoxia) that is commonly used in a laboratory. Despite the abundance of research regarding acute and chronic anoxic exposure and cold stress, the literature is lacking evidence linking anoxic stress with variable environmental conditions such as animal age and stress duration. We present novel ways to assess coma recovery and survival using readily available laboratory tools. Our findings suggest that younger age, exposure to colder temperatures and wet environments increase resistance to anoxic stress.

Cytokinin response factor 6 negatively regulates leaf senescence and is induced in response to cytokinin and numerous abiotic stresses.

Cytokinin response factor 6 (CRF6) is an Arabidopsis AP2/ERF transcription factor which is transcriptionally induced by cytokinin. Cytokinin is known to delay leaf senescence in wild-type (WT) plants, for example in dark-incubated detached leaves. This response is mediated by the cytokinin receptor Arabidopsis histidine kinase receptor 3 (AHK3). Similar to ahk3 mutants, crf6 leaves show decreased sensitivity to this cytokinin effect. Leaves overexpressing CRF6 retain more Chl than those of the WT under these conditions without exogenous cytokinin. It therefore appears that an increase in expression of CRF6 downstream of the perception of cytokinin by AHK3 is involved in the delay of leaf senescence. Intact crf6 plants also begin to undergo monocarpic senescence sooner than WT plants. Interestingly, plants overexpressing CRF6 display a more extreme acceleration of development than crf6 mutants, suggesting that a specific expression level or localization of CRF6 is necessary to prevent premature senescence. Expression analyses indicate that CRF6 is highly expressed in the veins of mature leaves and that this expression decreases with age. CRF6 expression is shown to be induced by abiotic stress, in addition to increased cytokinin. Together, these findings suggest that CRF6 functions to regulate developmental senescence negatively and may have a similar role in response to stress. CRF6 may therefore be involved in fine-tuning the timing of developmental and stress-induced senescence. CRF6 functioning in negative regulation of senescence is significant in that it is the first process known to be regulated by cytokinin, in which a CRF can be placed specifically downstream of the cytokinin signaling pathway.