DOI: 10.5061/dryad.7d7wm37vf
title: O-GlcNAc transferase OGT-1 and the ubiquitin ligase EEL-1 modulate seizure susceptibility in C. elegans Dataset

description: 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.
description: <strong><em>C.
 elegans</em></strong><strong> genetics and
 transgenics                                                      
 </strong> <em>Caenorhabditis
 elegans</em> used for the initial experiments were standard Bristol
 N2, loss-of-function <em>ogt (ok430)</em>, loss-of-function
 <em>eel-1 (bgg1)</em>, and the double loss-of-function
 <em>ogt-1(ok430); eel-1(bgg1)</em> strains. The
 <em>eel-1</em> protein null, <em>bggl</em>,
 deletes the entire <em>eel-1</em> coding sequence, including
 the HECT ubiquitin ligase domain. The
 <em>ogt-1</em>(<em>ok430</em>) mutant is a null
 allele with a large insertion/deletion that generates a frameshift
 upstream of the glycosyltransferase domain. For the <em>ogt-1;
 eel-1</em> double mutants, <em>eel-1</em> was balanced
 with <em>tmC25</em> [<em>tmIs1241</em>] to
 maintain the strain due to brood size and viability defects. WT animals
 and single mutants were also balanced as controls. Further rescue
 experiments were conducted using the <em>ogt-1(ok430)
 III;</em><em> eel-1(bgg1) IV; bggSi2[Prab-3::ogt-1a] II
 </em>strain. The Bristol N2 strain was ordered from the
 Caenorhabditis Genetics Center (NIH Office of Research Infrastructure
 Programs, P40OD010440). All <em>C. elegans</em> strains were
 maintained on standard NGM agar plates and seeded with 75μl of OP50
 <em>Escherichia coli</em>. All initial strains were plated 72
 hours prior to picking L4 stage <em>C. elegans </em>and
 incubated at 25°C. Approximately 24 hours prior to testing, the L4 worms
 were then picked and transferred to new NGM agar plates seeded with 75μl
 of OP50 <em>E. coli</em> and incubated overnight at
 25°C. <strong><em>C. elegans
 electroconvulsive</em></strong><strong> seizure
 assay                                         </strong>
 For the experiments in this
 study, wt , <em>eel-1 </em>and <em>ogt-1
 </em>mutants, and <em>ogt-1; eel-1</em> double mutants,
 and OGT-1 pan-neuronal rescue in<em> ogt-1; eel-1</em> double
 mutants were habituated in M9 solution for 30 minutes prior to electric
 shock. All <em>Caenorhabditis elegans </em>strains were run in
 the electroshock seizure assay as previously outlined [23, 35]. The
 experimental set-up included Grass SD9 stimulator, Grass SD44 stimulator
 (utilized as a 3 second timer), dissecting microscope with a camera
 (Hitachi model KP-D20BU), twelve-inch television monitor, and an HDD and
 DVR recorder (Magnavox model MDR535H/F7). In 10 mm long clear plastic
 tubing (Tygon<sup>® </sup>microbore tubing), 15μl of M9 saline
 solution was added. Roughly three to six 1-day-old adult worms were picked
 using a platinum wire pick and placed in those plastic tubes, after which
 they were incubated for 30 minutes. After incubation, an insulated copper
 electrode was inserted to both ends of the plastic tube. A 1cm gap was
 measured in between both electrodes. The electrodes were fastened with
 alligator clips to a square-pulse generating stimulator (Grass, SD9) that
 delivered a 3 second, 47V shock. Approximately six replicates per genotype
 were done. <strong><em>Electroconvulsive seizure assay video recovery time recording</em></strong><strong>     </strong> Video capture via a dissecting microscope camera (Hitachi model KP-D20BU) was initiated 10 seconds prior to the administration of the shock. Synchronized adults (L4) had a baseline behavior recorded for 10 seconds prior to administration of the electric shock. Speed was normalized to M9 buffer to examine the effects of a given genotype on locomotion. The mean speed was calculated every minute and corresponded to the total amount of sinusoidal wave-like swimming per worm. Video capture continued for up to 5 minutes after the shock had taken place. Due to electrolysis, bubbles formed on either end of the plastic tube. Data points corresponding to the recovery times of each <em>Caenorhabditis elegans</em> were then collected. Recovery time was defined as the point at which three normal sinusoidal wave-like swimming movement resumed following the initiation of the shock. Each individual worm was manually scored the minute they resumed sinusoidal motion. <em>C. elegans</em> were excluded from analysis if they intersected the peripheral bubbles. Those animals that did not recover after the shock were excluded from the time to recovery analysis and included in the percent of animals’ recovery analysis. All recovery times and percent of animals that recovered upon electroshock are shown as averages for each genotype. <strong><em>Pharmacological manipulations</em></strong><strong>                                                            </strong> Drugs were dissolved directly into M9 saline solution and 15μl of solution was aliquoted into transparent plastic tubing. Animals were incubated in drugs of interest for 30 minutes prior to electric shock. The drug tested was Baclofen and it was obtained from Sigma-Aldrich, St. Louis, MO, USA. <strong><em>Statistical analysis</em></strong><strong>                                                                                       </strong> Average time of recovery and average sinusoidal pre-shock movement data for wildtype, <em>eel-1 </em>null mutants, <em>ogt-1 </em>null mutants, <em>ogt-1; eel-1</em> double mutants and OGT-1 pan-neuronal rescue animals were analyzed using a One-Way Anova on Ranks with a Bonferroni Test - pairwise (Figs 1B and 2B).  Average time of recovery and average percent recovered for wildtype, <em>eel-1 </em>null mutants, <em>ogt-1 </em>null mutants, <em>ogt-1; eel-1</em> double mutants, and OGT-1 pan-neuronal rescue in <em>ogt-1; eel-1</em> double mutant strains were analyzed using a One-Way Anova on Ranks with a Student-Newman-Keuls Method-pairwise (Figs 2A and 2C). All bar graphs represent a mean ± SEM (error bars) and the letters denote statistical groupings. Significance between columns were denoted using a *P&lt;0.05, **P&lt;0.01, ***P&lt;0.001, and ns= not significant. Outliers greater than the 75th percentile value + 1.5*IQR and outliers less than the 25 percentile value - 1.5*IQR were removed.
description: Figure 1 and Figure 2 Dataset Excel Sheet: The sheets are labeled
 with the corresponding figure. Each column represents one genotype,
 labeled on the top of the column. The numbers in light red represents
 outliers, the numbers in red at the end of each column represent the
 average. To the right of each sheet is how outliers were determined to be
 removed. Figure 2C data indicates the survival, "100" denotes
 fully recovery after electroshock, "0" denotes no recovery after
 electroshock.  Figure 3 Dataset Excel Sheet with
 outliers removed: Each sheet is labeled with the experimental condition.
 Each column represents one genotype, labeled on the top of the
 column. Below each column are the averages and SEM. The dataset contains
 the data without the outliers.  Figure 3 Dataset with
 outliers: Each sheet is labeled with the experimental condition. Each
 column represents one genotype, labeled on the top of the column.  To the
 right of each sheet is how outliers were determined to be removed. The
 numbers in red represent outliers.