A Caenorhabditis elegans assay of seizure-like activity optimised for identifying antiepileptic drugs and their mechanisms of action

Wong, Shi Quan; Jones, Alistair; Dodd, Steven W.; Grimes, Douglas; Barclay, Jeff W.; Marson, Anthony G; Cunliffe, Vincent T.; Burgoyne, Robert D.; Sills, Graeme J.; Morgan, Alan

doi:10.1016/j.jneumeth.2018.09.004

Cited by 18 publications

(38 citation statements)

References 73 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To investigate whether the humanized strains displayed seizure‐like activity, we took advantage of a recently developed assay . This involves incubating worms with the proconvulsant PTZ and measuring the number of head‐bobbing convulsions produced.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Functional analysis of epilepsy‐associated variants in STXBP1/Munc18‐1 using humanizedCaenorhabditis elegans

et al. 2020

Self Cite

View full text Add to dashboard Cite

Objective: Genetic variants in STXBP1, which encodes the conserved exocytosis protein Munc18-1, are associated with a variety of infantile epilepsy syndromes. We aimed to develop an in vivo Caenorhabditis elegans model that could be used to test the pathogenicity of such variants in a cost-effective manner. Methods: The CRISPR/Cas9 method was used to introduce a null mutation into the unc-18 gene (the C. elegans orthologue of STXBP1), thereby creating a paralyzed worm strain. We subsequently rescued this strain with transgenes encoding the human STXBP1/Munc18-1 protein (wild-type and eight different epilepsy-associated missense variants). The resulting humanized worm strains were then analyzed via behavioral, electrophysiological, and biochemical approaches. Results: Transgenic expression of wild-type human STXBP1 protein fully rescued locomotion in both solid and liquid media to the same level as the standard wild-type worm strain, Bristol N2. Six variant strains (E59K, V84D, C180Y, R292H, L341P, R551C) exhibited impaired locomotion, whereas two (P335L, R406H) were no different from worms expressing wild-type STXBP1. Electrophysiological recordings revealed that all eight variant strains displayed less frequent and more irregular pharyngeal pumping in comparison to wild-type STXBP1-expressing strains. Four strains (V84D, C180Y, R292H, P335L) exhibited pentylenetetrazol-induced convulsions in an acute assay of seizure-like activity, in contrast to worms expressing wild-type STXBP1. No differences were seen between wild-type and variant STXBP1 strains in terms of mRNA abundance. However, STXBP1 protein levels were reduced to 20%-30% of wild-type in all variants, suggesting that the mutations result in STXBP1 protein instability. Significance: The approach described here is a cost-effective in vivo method for establishing the pathogenicity of genetic variants in STXBP1 and potentially other conserved neuronal proteins. Furthermore, the humanized strains we created could potentially be used in the future for high-throughput drug screens to identify novel therapeutics.

show abstract

Section: Resultsmentioning

confidence: 99%

“…This was performed using a recently described method . Briefly, worms were exposed to 7 mg/mL PTZ in Dent solution for 15 minutes and then head‐bobbing convulsions were stringently scored over 30 seconds.…”

Section: Methodsmentioning

confidence: 99%

Functional analysis of epilepsy‐associated variants in STXBP1/Munc18‐1 using humanizedCaenorhabditis elegans

et al. 2020

Self Cite

View full text Add to dashboard Cite

show abstract

“…21 PTZ and paralysis assays were performed as previously described. 21 The UBC_f80M224Q and CBGtg9050C11145D 27 fosmid constructs were obtained from Source-Bioscience. Three independently derived lines of each transgenic strain were analyzed.…”

Section: Elegans Methodsmentioning

confidence: 99%

“…7 Nevertheless, mutations in various C elegans genes have been shown to cause spontaneous seizurelike convulsions, or increased susceptibility to such behaviors, when exposed to PTZ, electroshock treatment, or heat, [17][18][19][20][21][22] consistent with higher organisms. Furthermore, various approved ASDs have been shown to ameliorate these seizurelike behaviors, 17,21 suggesting that C elegans has untapped potential for epilepsy drug discovery. In particular, the ease of genetic manipulation and maintenance of genetically modified strains in comparison to other animal models means that C elegans is especially powerful for discovering ASD mechanisms of action via chemical-genetic approaches.…”

Section: Introductionmentioning

confidence: 99%

“…In particular, the ease of genetic manipulation and maintenance of genetically modified strains in comparison to other animal models means that C elegans is especially powerful for discovering ASD mechanisms of action via chemical-genetic approaches. 21,23 Each model organism used in epilepsy research has its own strengths and weaknesses. We reasoned that combining the strengths of multiple in vivo animal models with in vitro brain tissue electrophysiology would create a pipeline approach for discovering novel ASDs and their mechanisms of action.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A multiorganism pipeline for antiseizure drug discovery: Identification of chlorothymol as a novel γ‐aminobutyric acidergic anticonvulsant

et al. 2020

Self Cite

View full text Add to dashboard Cite

show abstract

Functional analysis of epilepsy‐associated GABA_A receptor mutations using Caenorhabditis elegans

Gadhia,

Barker,

Morgan

et al. 2024

Epilepsia Open

Self Cite

View full text Add to dashboard Cite

ObjectiveGABAA receptor subunit mutations pose a significant risk for genetic generalized epilepsy; however, there are over 150 identified variants, many with unknown or unvalidated pathogenicity. We aimed to develop in vivo models for testing GABAA receptor variants using the model organism, Caenorhabditis elegans.MethodsCRISPR‐Cas9 gene editing was used to create a complete deletion of unc‐49, a C. elegans GABAA receptor, and to create homozygous epilepsy‐associated mutations in the endogenous unc‐49 gene. The unc‐49 deletion strain was rescued with transgenes for either the C. elegans unc‐49B subunit or the α1, β3, and γ2 subunits for the human GABAA receptor. All newly created strains were analyzed for phenotype and compared against existing unc‐49 mutations.ResultsNematodes with a full genetic deletion of the entire unc‐49 locus were compared with existing unc‐49 mutations in three separate phenotypic assays—coordinated locomotion, shrinker frequency and seizure‐like convulsions. The full unc‐49 deletion exhibited reduced locomotion and increased shrinker frequency and PTZ‐induced convulsions, but were not found to be phenotypically stronger than existing unc‐49 mutations. Rescue with the unc‐49B subunit or creation of humanized worms for the GABAA receptor both showed partial phenotypic rescue for all three phenotypes investigated. Finally, two epilepsy‐associated variants were analyzed and deemed to be loss of function, thus validating their pathogenicity.SignificanceThese findings establish C. elegans as a genetic model to investigate GABAA receptor mutations and delineate a platform for validating associated variants in any epilepsy‐associated gene.Plain Language SummaryEpilepsy is a complex human disease that can be caused by mutations in specific genes. Many possible mutations have been identified, but it is unknown for most of them whether they cause the disease. We tested the role of mutations in one specific gene using a small microscopic worm as an animal model. Our results establish this worm as a model for epilepsy and confirm that the two unknown mutations are likely to cause the disease.

show abstract

A Caenorhabditis elegans assay of seizure-like activity optimised for identifying antiepileptic drugs and their mechanisms of action

Abstract: HighlightsWorms with mutant GABAA receptors exhibit convulsions upon exposure to pentylenetetrazol.Convulsions are prevented by the approved anti-epileptic drug, ethosuximide.C. elegans model is a higher throughput, ethical alternative to rodent seizure models.

Cited by 18 publications

References 73 publications

Functional analysis of epilepsy‐associated variants in STXBP1/Munc18‐1 using humanizedCaenorhabditis elegans

Functional analysis of epilepsy‐associated variants in STXBP1/Munc18‐1 using humanizedCaenorhabditis elegans

A multiorganism pipeline for antiseizure drug discovery: Identification of chlorothymol as a novel γ‐aminobutyric acidergic anticonvulsant

Functional analysis of epilepsy‐associated GABA_A receptor mutations using Caenorhabditis elegans

Contact Info

Product

Resources

About

A Caenorhabditis elegans assay of seizure-like activity optimised for identifying antiepileptic drugs and their mechanisms of action

Abstract: HighlightsWorms with mutant GABAA receptors exhibit convulsions upon exposure to pentylenetetrazol.Convulsions are prevented by the approved anti-epileptic drug, ethosuximide.C. elegans model is a higher throughput, ethical alternative to rodent seizure models.

Cited by 18 publications

References 73 publications

Functional analysis of epilepsy‐associated variants in STXBP1/Munc18‐1 using humanizedCaenorhabditis elegans

Functional analysis of epilepsy‐associated variants in STXBP1/Munc18‐1 using humanizedCaenorhabditis elegans

A multiorganism pipeline for antiseizure drug discovery: Identification of chlorothymol as a novel γ‐aminobutyric acidergic anticonvulsant

Functional analysis of epilepsy‐associated GABAA receptor mutations using Caenorhabditis elegans

Contact Info

Product

Resources

About

Functional analysis of epilepsy‐associated GABA_A receptor mutations using Caenorhabditis elegans