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Effects of ASD-associated daf-18/PTEN missense variants on C. elegans dauer development

González-Cavazos, Carolina and Cao, Mengyi and Wong, Wan-Rong and Chai, Cynthia and Sternberg, Paul W. (2019) Effects of ASD-associated daf-18/PTEN missense variants on C. elegans dauer development. microPublication Biology . micropub.biology.000177. ISSN 2578-9430. PMCID PMC7252320. https://resolver.caltech.edu/CaltechAUTHORS:20200629-120228447

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Abstract

The relatively simple nervous system and facile genetics of Caenorhabditis elegans makes it a potential model organism to study the genetic basis of complex neural diseases, such as autism spectrum disorder (ASD). Our previously published study identified conserved human ASD-missense variants in C. elegans orthologs that have a role on morphology, locomotion or fecundity, suggesting C. elegans as an efficient phenotypic model to study conserved ASD variants (Wong et al., 2019). One of the ASD-associated genes screened in this study is daf-18, an ortholog of PTEN. DAF-18/PTEN is a lipid phosphatase protein that dephosphorylates PIP3, a critical lipid second messenger that mediates downstream kinase cascade signaling of the insulin pathway, that in turn regulates expression of genes involved in C. elegans lifespan and dauer formation (Murphy and Hu, 2013). It is reported that the daf-18(e1375) strain, which has a 30–base pairs insertion in the fourth exon, has a dauer defective phenotype (Ogg and Ruvkun, 1998); however, the effect of daf-18/PTEN single amino acid substitution in dauer formation has not been evaluated before. Here, we investigate the dauer entry abilities of previously published ASD-associated daf-18/PTEN missense variants (Wong et al., 2019). We performed a dauer-entry assay using crude pheromone as a proxy for high population density and heat-killed bacteria as a limited food source. We first determined the crude pheromone concentration required to induce 50% of wild type (N2) strain to enter dauer stage (EC50). Using the calculated EC50value we evaluated each mutant strain in two independent experiments (Table 1). To control day-to-day variations caused by environmental conditions, N2 controls were used in every trail. Additionally, we used the previously reported daf-18 dauer-defective strain (e1375) as a control (Ogg and Ruvkun, 1998). To analyze the obtained data, we performed one-way ANOVA followed by multiple comparisons test (Dunnett test) (Figure 1A). Table 1 Evaluated ASD-associated daf-18/PTEN mutant strains. Strain name PS7439 PS7432 PS7436 PS7430 PS7434 C. elegans allele name daf-18(sy879) daf-18(sy887) daf-18(sy881) daf-18(sy885) daf-18(sy882) C. elegans protein change D66E L115V H138R H168Q T176I Human protein change D22E L70V H93R H123Q T131I As expected, we observed statistically significant difference between our wild type and dauer defective control (e1375). Our results show that H138R (PS7436) and T176I (PS7434) daf-18 mutant strains have a significant difference from the wild type control, and a non-significant difference from the dauer defective control, suggesting these missense alleles cause dauer defective phenotypes to the same level of the e1375 strain. In contrast, we found the D66E (PS7439), L115V (PS7432) and H168Q (PS7430) mutant strains are significantly different from the dauer defective control and not significantly different from the wild type control, suggesting these three missense alleles do not cause the same level of dauer defective phenotype as the defective control does. Altogether, our current data show that H138R and T176I are defective in dauer entry, while D66E, L115V and H168Q daf-18 mutant strains are not (Figure 1A). The Conserved Domain Database (Conserved Domain Search) of NCBI (ID: G5EE01) predicts that four of the five missense variations evaluated in this study are located in the phosphatase domain of DAF-18/PTEN(Figure 1B). It is predicted that amino acid Histidine 138 is located in the active site of the protein, suggesting that H138R (PS7436) amino acid change might have an impact on PIP3 substrate specificity, which could explain the dauer defect phenotype observed. Moreover, it is predicted that both amino acid Histidine 168 and Threonine 176 are located close to the catalytic site of the phosphatase domain. We found a dauer defective phenotype resulting from a T176I substitution (PS7434) but not from a H168Q amino acid substitution (PS7430). These data suggest that certain amino acids in the catalytic and active sites of DAF-18/PTEN decrease the function of the protein, in turn regulating C. elegans dauer development. Future evaluation of PIP3 phosphorylation levels could confirm the effect of these variations on the phosphatase activity. The results of this study contribute to the knowledge of the mechanisms controlling C. elegans dauer entry, and to the C. elegans phenotype-screening of conserved ASD variations.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.17912/micropub.biology.000177DOIArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7252320PubMed CentralArticle
ORCID:
AuthorORCID
Sternberg, Paul W.0000-0002-7699-0173
Additional Information:© 2019 by the authors. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Received: September 30, 2019; Accepted: October 8, 2019; Published: October 16, 2019. Funding: CGC: Simons Foundation SFARI pilot grant and summer fellowship. MC: NIH-F32 fellowship (1 F32 GM131570-01). WRW, CC, PWS: NIH U01-NS111697. Author Contributions: Carolina González-Cavazos: Investigation, Formal analysis, Writing - original draft Mengyi Cao: Methodology, Formal analysis, Writing - original draft Wan-Rong Wong: Methodology, Writing - review and editing Cynthia Chai: Formal analysis, Writing - review and editing Paul W Sternberg: Conceptualization, Resources, Funding acquisition, Project administration, Writing - review and editing.
Funders:
Funding AgencyGrant Number
Simons FoundationUNSPECIFIED
NIH1 F32 GM131570-01
NIHU01-NS111697
PubMed Central ID:PMC7252320
Record Number:CaltechAUTHORS:20200629-120228447
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20200629-120228447
Official Citation:González-Cavazos, C; Cao, M; Wong, W; Chai, C; Sternberg, PW (2019). Effects of ASD-associated daf-18/PTEN missense variants on C. elegans dauer development. microPublication Biology. 10.17912/micropub.biology.000177
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:104128
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:29 Jun 2020 20:19
Last Modified:29 Jun 2020 20:19

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