Caenorhabditis elegans

Using the Caenorhabditis elegans as a model we have employed forward genetic screens to uncover several novel genetic contributors to dopamine (DA) signaling(1). Follow-up characterization of some of these novel contributors have been detailed in published work from our lab(2), while follow-up studies on other pathways are still underway. Moreover, using the powerful Million Mutation Project library, we have uncovered an important link between primary cilium formation and the regulation of the DA transporter dat-1(3). The focus of the body of work detailed in this manuscript is on a glial expressed gene, swip-10, uncovered from our original genetic screen(1, 4, 5). Unlike the other pathways uncovered from our genetic screening, swip-10 does not affect DA signaling via DAT-1 regulation, instead, loss of swip-10 produces excess DA signaling in a glutamate-signaling-dependent manner to cause swimming-induced paralysis (Swip)(4) as well as premature DA neuron degeneration(5). Specifically, the primary aim here was to uncover the molecular pathway by which swip-10 supports these phenotypes.

Cancer is a leading cause of death in the U.S and across the world, with estimates indicating 17 million new cancer cases in 2018, 9.5 million of which resulted in death. Statistics show that in the past 20 years cancer death rates have decreased 27% due to emerging therapies. The use of chemotherapies to kill fast-growing cells in the body has become one of the most common cancer treatments in the world today. Chemotherapy-Induced Peripheral Neuropathies (CIPNs) are the most common side effects caused by chemotherapeutic agents. CIPNs have a prevalence of up to 85% in cancer patients undergoing chemotherapy. CIPNs triggered by chemotherapeutic drug use severely damage nerves branching from either the brain or spinal cord, initiating the development of acute and/or chronic symptoms. Platinum-based and taxane-based chemotherapeutics are among the most potent and versatile drugs available for combating cancer. The two of these drugs, carboplatin and docetaxel, are known to cause peripheral neuropathies and central neurotoxicity and were the focus of this project.

Epilepsy is a prevalent brain disorder that affects more than 1 in 26 people in the United States. The recurring increased neuronal excitability during seizures results in sleep disturbances and muscle convulsions that reduce the quality of life and increase the healthcare costs for these patients. An epilepsy diagnosis is made when patients have had two or more seizures. There are many types of seizures and an individual can have more than one type. Seizures are classified into two groups, 1) generalized seizures that affect both sides of the brain and 2) focal seizures that are located in just one area of the brain. The causes of epilepsy vary by the age of the person, some with no clear cause may have a genetic form of epilepsy. Due to the various causes and types of seizures, many treatments including invasive surgeries and antiepileptic drugs (AEDs) do not work for all epileptic/seizure patients and are merely used to ease symptoms. The physiological complexity of the disorder and limited knowledge on its specific molecular mechanisms may contribute to the lack of effective treatment. In recent years, there has been an estimated average cost in billions of dollars to bring new medicine to the market; due to the lack of novel antiseizure targets and mechanism-based therapies on seizure phenotypes. In response to this, we utilized the electroconvulsive seizure behavioral assay to characterize one generalized seizure phenotype, tonic-clonic/grand mal seizures.

Over 70 million people worldwide suffer from epilepsy, with 90% of those cases taking place in developing countries (Singh & Trevick, 2016). Epilepsy can be defined as at least two unprovoked seizures occurring more than 24 hours apart, one unprovoked seizure with at least 60% chance of another seizure occurring within the next 10 years, or a diagnosis of epilepsy syndrome (Fisher et al., 2005). Varying physiological, molecular, genetic, and environmental factors can contribute to epileptic episodes. Although antiepileptic drugs (AEDs) exist, the complexity and lack of understanding behind the molecular mechanisms of the syndrome leaves the few drugs available to be insufficient for many patients (Rho & White, 2018). Therefore, the discovery of genetic pathways involved in epilepsy is imperative for the innovation of antiepileptic drugs. This thesis explores a novel method to add to mutant C.elegans libraries and improve antiepileptic drug discovery in a cost-effective and efficient manner by uncovering candidate molecular pathways through the candidate genes involved with antiepileptic strains.

Epilepsy is a widely prevalent disease within the United States. It is estimated that about 1.2% of the total American population has active epilepsy, a condition of the brain that causes seizures. These seizures are marked by chemical alterations in neuronal firing that can cause abnormal behavior, sensations, muscle spasms, and loss of consciousness. Although the prevalence of seizures and epilepsy is high, effective treatments are limited and fail to provide effective treatment for nearly one-third of adult epileptic patients. Here, I conclude results of successful screening of novel compounds that can ameliorate seizures using an electroshock assay to examine seizure susceptibility and duration in C. elegans. The use of this assay provides an excellent platform for novel antiepileptic drug (AED) discovery efficiently.
Literature shows Resveratrol, a natural product from plants, provides neuroprotective effects in various model organisms and therefore, is an excellent candidate for a molecule that has never been related to seizure. However, it is easily metabolized, being a flat and planar molecule. Our research group has collaboratively identified a novel bicyclic bridge molecule derived from the scaffolding of two resveratrol molecules we named Resveramorph (RVM). We also used the candidate approach to test a number of Resveramorph analogs on this assay to find the analog with highest efficacy. The various molecules characterized with their efficacy for seizure-like behavior after an electroshock have helped elucidate the mechanism of action and the RVMs physical target to give us greater insight into this potential family of AEDs.

Animals rely on the integration of a variety of external cues to understand and respond appropriately to their environment. The relative amounts of food and constitutively secreted pheromone detected by the nematode C. elegans determines how it will develop and grow. Starvation conditions cause the animal to enter a protective stage, termed dauer. Dauer animals are non-eating, long-lived and stress resistant. Yet, when these animals are introduced to food replete conditions they will recover from dauer and proceed into normal development. Furthermore, food restriction has been demonstrated to extend the lifespan of a wide-range of species including C. elegans. However, the exact mechanism by which food signals are detected and transduced by C. elegans to influence development and longevity remains unknown. Here, we identify a G protein-coupled receptor (GPCR) DCAR-1 that acts in two chemosensory neurons to mediate food signaling in an autophagy-related manner. The DCAR-1 ligand Dihydrocaffeic acid (DHCA) competes with dauer-inducing pheromone to promote growth. DHCA is a key intermediate in the shikimate pathway, which is required to synthesize folate and aromatic amino acids. We report that dcar-1 mutations influence dauer formation and extend wildtype lifespan via a mechanism of dietary restriction. Moreover, we show that the lifespan extension of dcar-1 mutants is completely dependent on autophagy gene atg- 18. Furthermore, our data suggests metabolites derived from shikimate are food signals that control aging and dauer development through GPCR signaling in C. elegans. These studies will contribute to the delineation of mechanisms behind the beneficial effects of dietary restriction in eukaryotic organisms.

Diseases such as epilepsy, pain, and neurodegenerative disorders are associated with changes in neuronal dysfunction due to an imbalance of excitation and inhibition. This work details a novel electroconvulsive seizure assay for C. elegans using the well characterized cholinergic and GABAergic excitation and inhibition of the body wall muscles and the resulting locomotion patterns to better understand neuronal excitability. The time to recover normal locomotion from an electroconvulsive seizure could be modulated by increasing and decreasing inhibition. GABAergic deficits and a chemical proconvulsant resulted in an increased recovery time while anti-epileptic drugs decreased seizure duration. Successful modulation of excitation and inhibition in the new assay led to the investigation of a cGMP-dependent protein kinase (PKG) which modulates potassium (K+) channels, affecting neuronal excitability, and determined that increasing PKG activity decreases the time to recovery from an electroconvulsive seizure. The new assay was used as a forward genetic screening tool using C. elegans and several potential genes that affect seizure susceptibility were found to take longer to recover from a seizure. A naturally occurring polymorphism for PKG in D. melanogaster confirmed that both genetic and pharmacological manipulation of PKG influences seizure duration. PKG has been implicated in stress tolerance, which can be affected by changes in neuronal excitability associated with aging, so stress tolerance and locomotor behavior in senescent flies was investigated. For the first time, PKG has been implicated in aging phenotypes with high levels of PKG resulting in reduced locomotion and lifespan in senescent flies. The results suggest a potential new role for PKG in seizure susceptibility and aging.

Seizures are a symptom of epilepsy, characterized by spontaneous firing due to an imbalance of excitatory and inhibitory features. While mammalian seizure models
receive the most attention, the simplicity and tractability of invertebrate model systems, specifically C. elegans and D. melanogaster, have many advantages in understanding
the molecular and cellular mechanisms of seizure behavior. This research explores C. elegans and D. melanogaster as electroconvulsive seizure models to investigate
methods to both modulate and better understand seizure susceptibility. A common underlying feature of seizures in mammals, worms, and flies involves regulating
excitation and inhibition. The C. elegans locomotor circuit is regulated via well characterized GABAergic and cholingeric motoneurons that innervate two rows of
dorsal and ventral body wall muscles. In this research, we developed an electroconvulsive seizure assay which utilizes the locomotor circuit as a behavioral read out of neuronal function. When inhibition is decreased in the circuit, for example by decreasing GABAergic input, we find a general increase in the time to recovery from a seizure. After establishing the contribution of excitation and inhibition to seizure recovery, we explored a ubiquitin ligase, associated with comorbidity of an X-linked Intellectual Disorder and epilepsy in humans, and established that the worm homolog, eel-1, contributes to seizure susceptibility similarly to the human gene. Next, we investigated a cGMP-dependent protein kinase (PKG) that functions in the nervous system of both worms and flies and determined that increasing PKG activity, decreases the time to recovery from an electroconvulsive seizure. These experiments suggest a potential novel role for a major protein, PKG, in seizure susceptibility and that the C. elegans and D. melanogaster electroconvulsive seizure assays can be used to investigate possible genes involved in seizure susceptibility and future therapeutic to treat epilepsy.

Several models for the evolution of maternal inheritance of mitochondria predict that the sperm mitochondria undergo oxidative damage and pose a threat to the developing embryo. Here, I test the hypothesis that the sperm are damaged by reactive oxygen species generated by aerobic sperm activity. In my first experiment, I found no significant difference in fecundity between worms fertilized by old versus young sperm, suggesting that the sperm nuclear genome is not affected by the extent of sperm activity. In my second experiment, I found that sperm mitochondrial DNA has deletions, indicating damage, but this damage does not accumulate with sperm activity. However, problems with PCR amplification resulted in little experimental data, preventing a conclusive test of the hypothesis.

Mitochondria are inherited uniparentally in almost all eukaryotic models studied to date. The fathers mitochondria are eliminated and there have been several hypothesis as to how this occurs. One hypothesis is that the sperm mitochondria are actively targeted and destroyed. Ubiquitin has been proposed a possible candidate involved in this process. My research investigated the normal mitochondrial inheritance pattern in C. elegans. I also examined the possible role of the C34F11.1 gene in mitochondrial inheritance. This gene is sperm-specific and has ubiquitin-ligase properties. It was determined that the normal mitochondrial inheritance pattern in C. elegans is maternal and that the sperm mitochondria are eliminated. It was also concluded that the C34F11.1 gene does not have a role in normal mitochondrial inheritance.