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. 2024 Apr 22:2024:10.17912/micropub.biology.001174.
doi: 10.17912/micropub.biology.001174. eCollection 2024.

Functional analysis of epilepsy-linked pathogenic variants of the Munc18-1 gene in the inhibitory nervous system of Caenorhabditis elegans

Keiko Gengyo-Ando  1   2 , Akane Osawa-Noguchi  2 , Hideki Ando  1 , Junichi Nakai  1   2
Affiliations

Functional analysis of epilepsy-linked pathogenic variants of the Munc18-1 gene in the inhibitory nervous system of Caenorhabditis elegans

Keiko Gengyo-Ando et al. MicroPubl Biol. .

Abstract

Heterozygous de novo mutations in Munc18-1, which is essential for neurotransmitter release, cause early infantile epileptic encephalopathy. Munc18-1-linked epilepsy is currently an untreatable disorder and its precise disease mechanism remains elusive. Here, we investigated how Munc18-1 pathogenic variants affect inhibitory neurons using Caenorhabditis elegans . Expression analysis revealed that three missense mutant proteins form aggregates in the cell body of gamma-aminobutyric-acid (GABA)-ergic motoneurons, resulting in a strong reduction of their expression in axons. Their defects of axonal expression correlated closely with pentylenetetrazol-induced convulsions, suggesting that the degree of instability of each mutant protein account for the severity of the epileptic phenotypes.

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Conflict of interest statement

The authors declare that there are no conflicts of interest present.

Figures

Figure 1. <b> Effects of <i>unc-18</i> mutations orthologous to pathogenic Munc18-1 mutations on expression of UNC-18 protein and GABAergic function in <i>C. elegans</i> </b>
Figure 1. Effects of unc-18 mutations orthologous to pathogenic Munc18-1 mutations on expression of UNC-18 protein and GABAergic function in C. elegans
(A) Three UNC-18 missense mutations analogous to pathogenic Munc18-1 mutations. Each mutated residue is shown in a space-filling representation in a 3D model of UNC-18. The Sec1/Munc18 family protein consists of three domains (domain 1: pink, domain 2: yellow, domain 3: blue). I84D is located in domain 1 and C178Y and G544D are located in domain 2. (B) Schematic diagram of a DNA construct for a 2A peptide (T2A)-mediated co-expression system. In this system, UNC-18::GFP and DsRed are theoretically expressed in a 1:1 molar ratio in GABAergic neurons. (C) Schematic representation of the GABAergic nervous system of C. elegans . (D) Expression patterns of the transgenic lines harboring the UNC-18 (WT)::GFP::T2A::DsRed (upper panels, jqEx248 ) and the UNC-18 (I84D)::GFP::T2A::DsRed (lower panels, jqEx252 ). WT proteins showed a soluble distribution pattern throughout the axons and the cell bodies (arrow) in the ventral nerve cord. In the mutant transgenic strain, DsRed was distributed normally in the ventral nerve cord and cell bodies, whereas I84D protein exhibited a punctate pattern (arrowheads) and a reduced expression in axons (brackets). Scale bars, 10 µm. (E) Aggregation of mutant UNC-18 proteins in the cell bodies of GABAergic neurons. In the transgenic lines expressing GFP-tagged mutant UNC-18, large aggregates (arrowheads) were observed. WT: jqEx248 , I84D: jqEx252 , C178Y: jqEx262 , G544D: jqEx258 . Scale bars, 5 µm. (F) Reduced expression of mutant UNC-18 proteins in the axons. WT proteins are distributed throughout the dorsal nerve cord (arrowheads), whereas mutant proteins are not detected (bracket). Dashed lines indicate the boundaries of the pharynx. Note that the signals in the intestine in the GFP channel are from the autofluorescence of the intestinal granules (Hermann et al., 2005). D: dorsal, V: ventral. Asterisks indicate coelomocyte RFP co-injection marker. Scale bars, 50 µm. (G) Quantification of percentage of GABAergic cell bodies with aggregates (n=98-110 from 11-13 animals). ***p<0.001. Statistics: Fisher's exact test, two-tailed. (H) Quantification of percentage of animals with no GFP signals in the dorsal nerve cord (n=101-113). ***p<0.001. Statistics: Fisher's exact test, two-tailed. (I) PTZ assay. The number of convulsion per minute were measured in unc-18(e81) , N2, WT: unc-18(e81);jqIs21 , I84D: unc-18(e81);jqIs23 , C178Y: unc-18(e81);jqIs9 , and G544D: unc-18(e81);jqIs43 . A higher PTZ index (the number of spasms per minute) represents more severe GABAergic defects. mean±SD. Statistics: one-way ANOVA followed by Tukey's multiple comparisons test. ***p<0.001.****p<0.0001. ns: not significant.
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Grants and funding

This work was supported in part by MEXT/JSPS KAKENHI Grant Number, JP17K07378, JP21K19693, JP21H05298 to KGA.

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