NHR-8 and P-glycoproteins uncouple xenobiotic resistance from longevity in chemosensory C. elegans mutants

  1. Gabriel A Guerrero
  2. Maxime J Derisbourg  Is a corresponding author
  3. Felix AMC Mayr
  4. Laura E Wester
  5. Marco Giorda
  6. J Eike Dinort
  7. Matías D Hartman
  8. Klara Schilling
  9. María José Alonso-De Gennaro
  10. Ryan J Lu
  11. Bérénice A Benayoun
  12. Martin S Denzel  Is a corresponding author
  1. Max Planck Institute for Biology of Ageing, Germany
  2. Leonard Davis School of Gerontology, University of Southern California, Los Angeles, United States
  3. CECAD - Cluster of Excellence University of Cologne, Germany
  4. Center for Molecular Medicine Cologne (CMMC), University of Cologne, Germany
5 figures, 1 table and 3 additional files

Figures

Figure 1 with 1 supplement
Tunicamycin resistance occurs in dye filling defective long-lived chemosensory C. elegans mutants.

(A) Fluorescence and differential interference contract confocal microscopy images of WT and osm-3(dh441)IV C. elegans after DiI treatment (scale bar 40 µm). (B) Depiction of the osm-3(dh441) locus. …

Figure 1—source data 1

Developmental TM resistance assay with WT and osm-3(dh441) animals (Figure 1C).

https://cdn.elifesciences.org/articles/53174/elife-53174-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Tunicamycin resistance occurs in dye filling defective long-lived chemosensory C. elegans mutants.

(A–J) Developmental TM resistance assay with WT, che-2(e1033)X, che-11(e1810)V, daf-10(e1387)V, dyf-1(mn335)I, osm-1(p808)X, osm-5(p813)X, osm-6(p811)V, odr-1(n1936)X, odr-3(n2150)V, and tax-4(p678)I…

Figure 1—figure supplement 1—source data 1

Developmental TM resistance assay with WT and che-2(e1033)X animals.

https://cdn.elifesciences.org/articles/53174/elife-53174-fig1-figsupp1-data1-v2.xlsx
Figure 1—figure supplement 1—source data 2

Developmental TM resistance assay with WT and che-11(e1810)V animals.

https://cdn.elifesciences.org/articles/53174/elife-53174-fig1-figsupp1-data2-v2.xlsx
Figure 1—figure supplement 1—source data 3

Developmental TM resistance assay with WT and daf-10(e1387)V animals.

https://cdn.elifesciences.org/articles/53174/elife-53174-fig1-figsupp1-data3-v2.xlsx
Figure 1—figure supplement 1—source data 4

Developmental TM resistance assay with WT and dyf-1(mn335)I animals.

https://cdn.elifesciences.org/articles/53174/elife-53174-fig1-figsupp1-data4-v2.xlsx
Figure 1—figure supplement 1—source data 5

Developmental TM resistance assay with WT and osm-1(p808)X animals.

https://cdn.elifesciences.org/articles/53174/elife-53174-fig1-figsupp1-data5-v2.xlsx
Figure 1—figure supplement 1—source data 6

Developmental TM resistance assay with WT and osm-5(p813)X animals.

https://cdn.elifesciences.org/articles/53174/elife-53174-fig1-figsupp1-data6-v2.xlsx
Figure 1—figure supplement 1—source data 7

Developmental TM resistance assay with WT and osm-6(p811)V animals.

https://cdn.elifesciences.org/articles/53174/elife-53174-fig1-figsupp1-data7-v2.xlsx
Figure 1—figure supplement 1—source data 8

Developmental TM resistance assay with WT and odr-1(n1936)X animals.

https://cdn.elifesciences.org/articles/53174/elife-53174-fig1-figsupp1-data8-v2.xlsx
Figure 1—figure supplement 1—source data 9

Developmental TM resistance assay with WT and odr-3(n2150)V animals.

https://cdn.elifesciences.org/articles/53174/elife-53174-fig1-figsupp1-data9-v2.xlsx
Figure 1—figure supplement 1—source data 10

Developmental TM resistance assay with WT and tax-4(p7689)III animals.

https://cdn.elifesciences.org/articles/53174/elife-53174-fig1-figsupp1-data10-v2.xlsx
Figure 2 with 1 supplement
Reduced tunicamycin-induced ER stress signaling in osm-3 mutants despite functional ER-UPR pathway.

(A) DAVID gene ontology (GO) terms that are enriched in TM-treated WT compared to TM-treated osm-3(dh441)IV worms. Red = upregulated and blue = downregulated. The fold enrichment is plotted as x …

Figure 2—source data 1

Biosorter analysis of osm-3(dh441)IV vs. WT animals in the hsp-4::GFP background after 6 hr of control or TM treatment (Figure 2C).

https://cdn.elifesciences.org/articles/53174/elife-53174-fig2-data1-v2.xlsx
Figure 2—source data 2

Biosorter analysis of osm-3(dh441)IV vs. WT animals in the hsp-4::GFP background raised on control or sel-1 RNAi.

https://cdn.elifesciences.org/articles/53174/elife-53174-fig2-data2-v2.xlsx
Figure 2—source data 3

Quantitative PCR measuring relative hsp-4 and spliced and unspliced xbp-1 mRNA levels in WT and osm-3(dh441)IV animals after 2 hr of 10 mM DTT treatment (Figure 2F).

https://cdn.elifesciences.org/articles/53174/elife-53174-fig2-data3-v2.xlsx
Figure 2—figure supplement 1
Quantitative PCR measuring relative mRNA expression of indicated genes in WT animals and osm-3(dh441)IV mutants after 6 hr of vehicle or TM treatment.

Fold change represents mRNA expression levels of the targeted genes normalized to WT treated with vehicle. Data are mean + SEM, n = 2.

Figure 2—figure supplement 1—source data 1

Quantitative PCR measuring relative mRNA expression of indicated genes in WT animals and osm-3(dh441)IV mutants after 6 hr of vehicle or TM treatment.

https://cdn.elifesciences.org/articles/53174/elife-53174-fig2-figsupp1-data1-v2.xlsx
Figure 3 with 1 supplement
Tunicamycin resistance in osm-3 mutants is not daf-16 or pmk-1 dependent.

(A) Demographic lifespan analysis of WT, osm-3(dhh441)IV, daf-16(mu86)I and osm-3(dh441)IV; daf-16(mu86)I animals. WT mean lifespan = 22 days, osm-3(dh441)IV mean lifespan = 25 days p<0.005 compared …

Figure 3—source data 1

Developmental resistance assay using 10 µg/mL TM with WT, osm-3(dh441)IV, daf-16(mu86)I, and osm-3(dh441)IV; daf-16(mu86)I mutants.

https://cdn.elifesciences.org/articles/53174/elife-53174-fig3-data1-v2.xlsx
Figure 3—source data 2

Developmental resistance assay using 10 µg/mL TM with WT, osm-3(dh441)IV, pmk-1(km25)IV, and osm-3(dh441)IV; pmk-1(km25)IV mutants.

https://cdn.elifesciences.org/articles/53174/elife-53174-fig3-data2-v2.xlsx
Figure 3—figure supplement 1
Tunicamycin resistance in osm-3 mutants is not daf-16 or pmk-1 dependent.

(A) Biosorter analysis of osm-3(dh441)IV vs. WT animals in the hsp-4::GFP background raised on control or daf-16 RNAi. Data are mean + SEM, n = 4, ***p<0.0001 by two-way ANOVA. (B) Developmental …

Figure 3—figure supplement 1—source data 1

Biosorter analysis of osm-3(dh441)IV vs. WT animals in the hsp-4::GFP background raised on control or daf-16 RNAi.

https://cdn.elifesciences.org/articles/53174/elife-53174-fig3-figsupp1-data1-v2.xlsx
Figure 3—figure supplement 1—source data 2

Developmental resistance assay using 10 µg/mL TM with WT, osm-3(dh441) IV, and daf-2(e1370) III mutants.

https://cdn.elifesciences.org/articles/53174/elife-53174-fig3-figsupp1-data2-v2.xlsx
Figure 4 with 1 supplement
nhr-8 signaling regulates xenobiotic detoxification response through PGPs.

(A) Quantitative PCR measuring relative PGP mRNA expression in WT, osm-3(dh441)IV, nhr-8(hd117)IV, and osm-3(dh441)IV; nhr-8(hd117)IV animals. Relative mRNA expression is mRNA expression levels …

Figure 4—source data 1

Quantitative PCR measuring relative PGP mRNA expression in WT, osm-3(dh441)IV, nhr-8(hd117)IV, and osm-3(dh441)IV; nhr-8(hd117)IV animals (Figure 4A).

https://cdn.elifesciences.org/articles/53174/elife-53174-fig4-data1-v2.xlsx
Figure 4—source data 2

Developmental TM resistance assay using 10 µg/mL TM and the indicated PGPs RNAi treatment.

https://cdn.elifesciences.org/articles/53174/elife-53174-fig4-data2-v2.xlsx
Figure 4—source data 3

Developmental TM resistance assay using the PGP inhibitor verapamil (VPL) using 10 µg/mL TM supplemented with vehicle or 1 nM VPL.

https://cdn.elifesciences.org/articles/53174/elife-53174-fig4-data3-v2.xlsx
Figure 4—source data 4

Developmental TM resistance assay on 10 µg/mL TM and control with WT, osm-3(dh441)IV, nhr-8(hd117)IV and osm-3(dh441)IV; nhr-8(hd117)IV animals (Figure 4D).

https://cdn.elifesciences.org/articles/53174/elife-53174-fig4-data4-v2.xlsx
Figure 4—source data 5

Developmental ivermectin resistance assay of WT, osm-3(dh441)IV, nhr-8(hd117)IV and osm-3(dh441)IV; nhr-8(hd117)IV animals using 6 ng/mL ivermectin and vehicle control (Figure 4E).

https://cdn.elifesciences.org/articles/53174/elife-53174-fig4-data5-v2.xlsx
Figure 4—source data 6

Developmental paraquat resistance assay of WT, osm-3(dh441)IV, nhr-8(hd117)IV, and osm-3(dh441)IV; nhr-8(hd117)IV animals using 0.2 mM paraquat and vehicle control (Figure 4F).

https://cdn.elifesciences.org/articles/53174/elife-53174-fig4-data6-v2.xlsx
Figure 4—figure supplement 1
nhr-8 signaling regulates xenobiotic detoxification response through PGPs.

(A) Heat shock assay at 35°C in WT and osm-3(dh441)IV animals on day 1 of adulthood. See Supplementary file 1 for statistical analysis. (B) Hydrogen peroxide survival assay using 1 µM H2O2 on day 1 …

Figure 4—figure supplement 1—source data 1

Heat shock assay at 35 °C in WT and osm-3(dh441)IV animals on day 1 of adulthood.

https://cdn.elifesciences.org/articles/53174/elife-53174-fig4-figsupp1-data1-v2.xlsx
Figure 4—figure supplement 1—source data 2

Hydrogen peroxide survival assay using 1 mM H2O2 on day 1 of adulthood in WT and osm-3(dh441)IV animals.

https://cdn.elifesciences.org/articles/53174/elife-53174-fig4-figsupp1-data2-v2.xlsx
Figure 4—figure supplement 1—source data 3

WT development on 0–1 µM Verapamil.

https://cdn.elifesciences.org/articles/53174/elife-53174-fig4-figsupp1-data3-v2.xlsx
Cholesterol modulates TM resistance through NHR-8.

(A) Developmental dose–response TM resistance assay using the indicated TM concentrations with WT animals and osm-3(dh441)IV mutants raised on 0 or 5 µg/mL cholesterol. Data are mean ± SD (n = 5 …

Figure 5—source data 1

Developmental dose–response TM resistance assay using the indicated TM concentrations with WT animals and osm-3(dh441)IV mutants raised on 0 or 5 µg/mL cholesterol.

https://cdn.elifesciences.org/articles/53174/elife-53174-fig5-data1-v2.xlsx
Figure 5—source data 2

Biosorter analysis of WT animals with the hsp-4::GFP reporter raised on 0 or 50 µg/mL cholesterol after 6 hr of vehicle or 5 µg/mL TM treatment.

https://cdn.elifesciences.org/articles/53174/elife-53174-fig5-data2-v2.xlsx
Figure 5—source data 3

Cholesterol dose–response developmental assay with WT, osm-3(dh441)IV, nhr-8(hd117)IV, and osm-3(dh441)IV; nhr-8(hd117)IV animals on vehicle or 5 µg/mL TM.

https://cdn.elifesciences.org/articles/53174/elife-53174-fig5-data3-v2.xlsx

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background(E. coli)HT115 [L4440::sel-1]Source BioscienceCELE_F45D3.5Ahringer RNAi library
Strain, strain background(E. coli)HT115 [L4440::daf-16]Source BioscienceCELE_R13H8.1Ahringer RNAi library
Strain, strain background(E. coli)HT115 [L4440::pgp-5]Source BioscienceCELE_C05A9.1Ahringer RNAi library
Strain, strain background(E. coli)HT115 [L4440::pgp-8]Source BioscienceCELE_T21E8.3Ahringer RNAi library
Strain, strain background(E. coli)HT115 [L4440::pgp11]Source BioscienceCELE_DH11.3Ahringer RNAi library
Strain, strain background(E. coli)HT115 [L4440::pgp-12]Source BioscienceCELE_F22E10.1Ahringer RNAi library
Strain, strain background(E. coli)HT115 [L4440::pgp-13]Source BioscienceCELE_F22E10.2Ahringer RNAi library
Genetic reagent(C. elegans)osm-3(dh441)IVOtherAA1962available from A. Antebi or M.S. Denzel
Genetic reagent(C. elegans)daf-16(mu86)ICGCCF1038
Genetic reagent(C. elegans)pmk-1(km25)IVCGCKU25
Genetic reagent(C. elegans)osm-3(dh441)IV; nhr-8(hd117)IVThis paperMSD420available from M.S. Denzel
Genetic reagent(C. elegans)osm-3(dh441)IV; daf-16(mu86)IThis paperMSD422available from M.S. Denzel
Genetic reagent(C. elegans)osm-3(dh441)IV; pmk-1(km25)IVThis paperMSD423available from M.S. Denzel
Genetic reagent(C. elegans)odr-1(n1936)XCGCCX2065
Genetic reagent(C. elegans)odr-3(n2150)VCGCCX2205
Genetic reagent(C. elegans)osm-6(p811)VCGCPR811
Genetic reagent(C. elegans)tax-4(p678)IIICGCPR678
Genetic reagent(C. elegans)nhr-8(hd117)IVotherAA968available from A. Antebi or M.S. Denzel
Genetic reagent(C. elegans)N2; dhEx451(nhr-8::gfp; coel::RFP)CGCAA1027
Genetic reagent(C. elegans)osm-3(dh441)IV; zcIs4 [hsp-4::GFP]VotherAA2774available from A. Antebi or M.S. Denzel
Genetic reagent(C. elegans)zcIs4[hsp-4::GFP]VCGCSJ4005
Genetic reagent(C. elegans)che-11(e1810)VCGCCB3330
Genetic reagent(C. elegans)daf-10(e1387)IVCGCCB1387
Genetic reagent(C. elegans)osm-1(p808)XCGCPR808
Genetic reagent(C. elegans)dyf-1(mn335)ICGCSP1205
Genetic reagent(C. elegans)che-2(e1033)XCGCCB1033
Genetic reagent(C. elegans)osm-5(p813)XCGCPR813
Commercial assay or kitDirect-zol RNA MicroprepZymo ResearchR2060
Commercial assay or kitSMARTer Stranded RNA-Seq HT KitTakara634,838
Commercial assay or kitRibo-zero Gold kitIlluminaMRZG12324
Chemical compound, drugTunicamycinCalbiochemCAS 11089-65-9
Chemical compound, drugDithiothreitolSigma-AldrichCAS 3483-12-3
Chemical compound, drugMethyl viologen dichloride hydrateSigma-AldrichCAS 75365-73-0Paraquat
Chemical compound, drugIvermectinSigma-AldrichCAS 70288-86-7
Chemical compound, drugVerapamil hydrochlorideSigma-AldrichCAS 152-11-4
Chemical compound, drugCholesterolSigma-AldrichCAS 57-88-5
Chemical compound, drug1,1′-Dioctadecyl-3,3,3′,3′-
tetramethylindocarbo
cyanine perchlorate
Sigma-AldrichCAS 41085-99-8DiI
Software, algorithmR statistical softwarecran.r-project.orgDESeq2 1.16.1PMID.25516281
OtherReference GenomeENSEMBLWBCel235

Additional files

Supplementary file 1

Table containing the statistical analysis of the lifespan, PA14, heat shock and H2O2 resistance assays.

https://cdn.elifesciences.org/articles/53174/elife-53174-supp1-v2.docx
Supplementary file 2

List of qPCR primers used in this study.

https://cdn.elifesciences.org/articles/53174/elife-53174-supp2-v2.docx
Transparent reporting form
https://cdn.elifesciences.org/articles/53174/elife-53174-transrepform1-v2.pdf

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