Ken Dawson-Scully

Neurodevelopmental disorders such as epilepsy and autism have been linked to an imbalance of excitation and inhibition (E/I) in the central nervous system. The simplicity and tractability of C. elegans allows our electroconvulsive seizure (ES) assay to be used as a behavioral readout of the locomotor circuit and neuronal function. C. elegans possess conserved nervous system features such as gamma-aminobutyric acid (GABA) and GABA receptors in inhibitory neurotransmission, and acetylcholine (Ach) and acetylcholine receptors in excitatory neurotransmission. Our previously published data has shown that decreasing inhibition in the motor circuit, via GABAergic manipulation, will extend the time of locomotor recovery following electroshock. Similarly, mutations in a HECT E3 ubiquitin ligase called EEL-1 leads to impaired GABAergic transmission, E/I imbalance and altered sensitivity to electroshock. Mutations in the human ortholog of EEL-1, called HUWE1, are associated with both syndromic and non-syndromic intellectual disability. Both EEL-1 and its previously established binding protein, OGT-1, are expressed in GABAergic motor neurons, localize to GABAergic presynaptic terminals, and function in parallel to regulate GABA neuron function. In this study, we tested behavioral responses to electroshock in wildtype, ogt-1, eel-1 and ogt-1; eel-1 double mutants. Both ogt-1 and eel-1 null mutants have decreased inhibitory GABAergic neuron function and increased electroshock sensitivity. Consistent with EEL-1 and OGT-1 functioning in parallel pathways, ogt-1; eel-1 double mutants showed enhanced electroshock susceptibility. Expression of OGT-1 in the C. elegans nervous system rescued enhanced electroshock defects in ogt-1; eel-1 double mutants. Application of a GABA agonist, Baclofen, decreased electroshock susceptibility in all animals. Our C. elegans electroconvulsive seizure assay was the first to model a human X-linked Intellectual Disability (XLID) associated with epilepsy and suggests a potential novel role for the OGT-1/EEL-1 complex in seizure susceptibility.

The common fruit fly, Drosophila melanogaster, is a well-characterized model for neurological disorders and is widely used to investigate the biology of aging, stress tolerance, and pleiotropy. The foraging (for) gene encodes a cGMP-dependent protein kinase (PKG), which has been implicated in several behavioral phenotypes including feeding, sleep, learning and memory, and environmental stress tolerance. We used the well-established Drosophila Activity Monitor (DAM) to investigate the effects of the conserved NO/cGMP/PKG signaling pathway on functional senescence. Our results show that the polymorphic for gene confers protection during low oxygen stress at the expense of longevity and a decline in locomotor activity with age in D. melanogaster which suggests a novel role for the PKG pathway in healthy aging and senescence.

A cGMP-dependent protein kinase (PKG) has previously been shown to regulate synaptic transmission at the Drosophila neuromuscular junction (NMJ) during acute oxidative stress, potentially through modulation of downstream K⁺ channel kinetics; however, the specific K⁺ channels through which PKG functions remains unclear. In this study, we hypothesized that PKG may be acting on calcium-activated large-conductance Slo K⁺ channels, or BK channels. We found that genetic elimination and pharmacological inhibition of BK channel conductance increases synaptic transmission tolerance to acute H₂O₂-induced oxidative stress. Furthermore, we discovered that activation of PKG in BK channel loss-of-function (Slo⁴) mutants significantly decreases time to stimulus-induced synaptic failure, providing the first evidence of PKG and BK channels functioning independently to control synaptic transmission tolerance to acute oxidative stress.

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.

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), which 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.

The nematode Caenorhabditis elegans is a useful model organism for dissecting molecular mechanisms of neurological diseases. While hermaphrodite C. elegans contain 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.

While the mammalian brain functions within a very narrow range of oxygen concentrations and temperatures, the fruit fly, Drosophila melanogaster, has employed strategies to deal with a much wider range of acute environmental stressors. The foraging (for) gene encodes the cGMP-dependent protein kinase (PKG), has been shown to regulate thermotolerance in many stress-adapted species, including Drosophila, and could be a potential therapeutic target in the treatment of hyperthermia in mammals. Whereas previous thermotolerance studies have looked at the effects of PKG variation on Drosophila behavior or excitatory postsynaptic potentials at the neuromuscular junction (NMJ), little is known about PKG effects on presynaptic mechanisms. In this study, we characterize presynaptic calcium ([Ca²⁺]_i) dynamics at the Drosophila larval NMJ to determine the effects of high temperature stress on synaptic transmission. We investigated the neuroprotective role of PKG modulation both genetically using RNA interference (RNAi), and pharmacologically, to determine if and how PKG affects presynaptic [Ca²⁺]_i dynamics during hyperthermia. We found that PKG activity modulates presynaptic neuronal Ca²⁺ responses during acute hyperthermia, where PKG activation makes neurons more sensitive to temperature-induced failure of Ca²⁺ flux and PKG inhibition confers thermotolerance and maintains normal Ca²⁺ dynamics under the same conditions. Targeted motoneuronal knockdown of PKG using RNAi demonstrated that decreased PKG expression was sufficient to confer thermoprotection. These results demonstrate that the PKG pathway regulates presynaptic motoneuronal Ca²⁺ signaling to influence thermotolerance of presynaptic function during acute hyperthermia.

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 GABA_A 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 GABA_A receptor antagonist and proconvulsant in mammalian and C. elegans seizure models, increases susceptibility of worms to electric shock.

Natural products have provided an invaluable source of inspiration in the drug discovery pipeline. The oceans are a vast source of biological and chemical diversity. Recently, this untapped resource has been gaining attention in the search for novel structures and development of new classes of therapeutic agents. Pseudopterosins are group of marine diterpene glycosides that possess an array of potent biological activities in several therapeutic areas. Few studies have examined pseudopterosin effects during cellular stress and, to our knowledge, no studies have explored their ability to protect synaptic function. The present study probes pseudopterosin A (PsA) for its neuromodulatory properties during oxidative stress using the fruit fly, Drosophila melanogaster. We demonstrate that oxidative stress rapidly reduces neuronal activity, resulting in the loss of neurotransmission at a well-characterized invertebrate synapse. PsA mitigates this effect and promotes functional tolerance during oxidative stress by prolonging synaptic transmission in a mechanism that differs from scavenging activity. Furthermore, the distribution of PsA within mammalian biological tissues following single intravenous injection was investigated using a validated bioanalytical method. Comparable exposure of PsA in the mouse brain and plasma indicated good distribution of PsA in the brain, suggesting its potential as a novel neuromodulatory agent.