O-GlcNAc transferase OGT-1 and the ubiquitin ligase EEL-1 modulate seizure susceptibility in C. elegans Dataset
Data files
Dec 16, 2022 version files 52.96 KB
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Figure_3_Dataset_with_outliers_.xlsx
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Figure_3_Dataset_with_outliers_removed.xlsx
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Figures_1_and_2_Dataset_.xlsx
Abstract
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.
Methods
C. elegans genetics and transgenics
Caenorhabditis elegans used for the initial experiments were standard Bristol N2, loss-of-function ogt (ok430), loss-of-function eel-1 (bgg1), and the double loss-of-function ogt-1(ok430); eel-1(bgg1) strains. The eel-1 protein null, bggl, deletes the entire eel-1 coding sequence, including the HECT ubiquitin ligase domain. The ogt-1(ok430) mutant is a null allele with a large insertion/deletion that generates a frameshift upstream of the glycosyltransferase domain. For the ogt-1; eel-1 double mutants, eel-1 was balanced with tmC25 [tmIs1241] 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 ogt-1(ok430) III; eel-1(bgg1) IV; bggSi2[Prab-3::ogt-1a] II strain. The Bristol N2 strain was ordered from the Caenorhabditis Genetics Center (NIH Office of Research Infrastructure Programs, P40OD010440). All C. elegans strains were maintained on standard NGM agar plates and seeded with 75μl of OP50 Escherichia coli. All initial strains were plated 72 hours prior to picking L4 stage C. elegans 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 E. coli and incubated overnight at 25°C.
C. elegans electroconvulsive seizure assay
For the experiments in this study, wt , eel-1 and ogt-1 mutants, and ogt-1; eel-1 double mutants, and OGT-1 pan-neuronal rescue in ogt-1; eel-1 double mutants were habituated in M9 solution for 30 minutes prior to electric shock. All Caenorhabditis elegans 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® 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.
Electroconvulsive seizure assay video recovery time recording
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 Caenorhabditis elegans 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. C. elegans 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.
Pharmacological manipulations
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.
Statistical analysis
Average time of recovery and average sinusoidal pre-shock movement data for wildtype, eel-1 null mutants, ogt-1 null mutants, ogt-1; eel-1 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, eel-1 null mutants, ogt-1 null mutants, ogt-1; eel-1 double mutants, and OGT-1 pan-neuronal rescue in ogt-1; eel-1 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<0.05, **P<0.01, ***P<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.
Usage notes
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.