Neuronal TORC1 modulates longevity via AMPK and cell nonautonomous regulation of mitochondrial dynamics in C. elegans
- Harvard T. H. Chan School of Public Health, United States
- National Cheng Kung University, Taiwan
- Université Côte d’Azur, CNRS, INSERM, IRCAN, France
Figures
Figure 1 with 1 supplement
Neuronal AMPK is required for TORC1-mediated longevity.
(a, b) AMPK T172 phosphorylation is increased in raga-1(ok386) null mutants compared to wild type animals. Actin levels are used as loading controls (representative immunoblot and corresponding …
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Figure 1—source data 1
Figure 1b AMPK activity is increased in raga-1 mutants.
- https://doi.org/10.7554/eLife.49158.004
Figure 1—figure supplement 1
Conservation of critical residues in AAK-2 and their requirement for TORC1-mediated longevity.
(a) Western blot showing that a threonine-to-alanine mutation at T243 generated by CRISPR on aak-2 completely loses recognition for antibodies against phosphorylation at the conserved residue T172 …
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Figure 1—figure supplement 1—source data 1
Figure 1—figure supplement 1c AMPK activity is increased by knockdown of TOR.
- https://doi.org/10.7554/eLife.49158.005
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Figure 1—figure supplement 1—source data 2
Figure 1—figure supplement 1f The conserved S6K/Akt phosphorylation site serine 551 on AAK-2 modulates AMPK activity.
- https://doi.org/10.7554/eLife.49158.006
Figure 2 with 3 supplements
TORC1 signaling is required in neurons to regulate lifespan.
(a) The raga-1(ok386) deletion increases lifespan (p<0.0001). However, when raga-1 is expressed in the nervous system via extrachromosomal array using the rab-3 promoter, raga-1(ok386) does not …
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Figure 2—source data 1
Figure 2e qPCR of raga-1 expression in SCIs and extrachromosomal lines.
- https://doi.org/10.7554/eLife.49158.011
Figure 2—figure supplement 1
Neuronal raga-1 expressed by extrachromosomal transgene suppresses raga-1 mutant longevity when animals are not treated with FUDR to prevent progeny development.
Details on strains and lifespan replicates can be found in Supplementary file 6.
Figure 2—figure supplement 2
Elimination of raga-1 in the intestine by RNAi does not impair rescue by the extrachromosomal neuronal raga-1 array.
(a) raga-1 RNAi eliminates non-specific expression of the rab-3p::raga-1::SL2::mCherry extrachromosomal transgene in the intestine but preserves expression in neurons. Images were taken from adult …
Figure 2—figure supplement 3
Neuronal raga-1 does not rescue development delay of raga-1(ok386) mutants.
Stacked bar graph showing percent of animals at the noted developmental stage after 72 hr at 20°C. Shown are averaged values from two independent experiments. Error bars denote SEM. ‘neuronal raga-1’…
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Figure 2—figure supplement 3—source data 1
Figure 2—figure supplement 3 Developmental stages of raga-1 rescue lines.
- https://doi.org/10.7554/eLife.49158.012
Figure 3 with 3 supplements
Neuronal TORC1 modulates aging via changes to organelle organization and neuropeptide signaling.
(a) Cluster analysis identified 59 genes that show increased expression in raga-1(ok386) that is reversed by neuronal rescue array (Cluster 3, top) and 107 genes that show decreased expression in rag…
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Figure 3—source data 1
Figure 3c and d qPCR of daf-28 and ins-6.
- https://doi.org/10.7554/eLife.49158.017
Figure 3—figure supplement 1
Gene clusters and differentially represented GO terms identified by analysis of RNA-seq.
(a) Plot depicting the profiles of gene expression defining each cluster of differentially expressed genes. Most differentially expressed genes in raga-1(ok386) are not rescued by the neuronal raga-1…
Figure 3—figure supplement 2
Validation of changes identified by RNA-seq in independent biological samples.
Independent biological samples were collected and tested for gene expression changes identified by RNA-Seq by qPCR. Three new samples for each strain used in the RNA-Seq experiment (wild type (N2), r…
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Figure 3—figure supplement 2—source data 1
Figure 3—figure supplement 2 qPCR validation of RNA seq results.
- https://doi.org/10.7554/eLife.49158.018
Figure 3—figure supplement 3
Neuronal raga-1 regulates ins-6 expression in adults.
Relative expression of ins-6 in animals at day 1 of adulthood as determined by qPCR. Points plotted are from independent biological samples. Error bars denote mean + /- SEM. P values determined by …
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Figure 3—figure supplement 3—source data 1
Figure 3—figure supplement 3 qPCR of ins-6 in day 1 adults.
- https://doi.org/10.7554/eLife.49158.019
Figure 4 with 6 supplements
Neuronal RAGA-1 drives mitochondrial fragmentation in muscle cells.
(a) Representative pictures showing that loss of raga-1 preserves muscle mitochondrial content during aging, while neuronal RAGA-1 reverses these effects as seen in the corresponding skeletonized …
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Figure 4—source data 1
Figure 4c-h and Figure 4—figure supplement 5 Effects of neuronal raga-1 rescue on parameters of muscle mitochondrial morphology.
- https://doi.org/10.7554/eLife.49158.027
Figure 4—figure supplement 1
Workflow for MitoMAPR analysis.
A Region of Interest (15 × 15 um) was processed by the MitoMAPR macro. The ROI is enhanced for signal intensity by Enhance Local Contrast (CLAHE) followed by conversion to Binary. The binary image …
Figure 4—figure supplement 2
Examples of networks analyzed by MitoMAPR.
Using Networks (N) and Junction Point (JP) values, it is possible to estimate the degree of complexity of the mitochondrial network. As illustrated the four cells (A–D) have their mitochondria …
Figure 4—figure supplement 3
raga-1 deletion affects mitochondrial network states in neurons.
(a–b) Representative pictures (a) showing that loss of raga-1 preserves mitochondrial content during aging, in neurons as seen in the overlay images (Left) post MitoMAPR processing. The …
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Figure 4—figure supplement 3—source data 1
Figure 4—figure supplement 3c-f Effects of neuronal raga-1 rescue on parameters of neuronal mitochondrial morphology.
- https://doi.org/10.7554/eLife.49158.028
Figure 4—figure supplement 4
raga-1 deletion prevents mitochondria fragmentation in intestine.
(a) Mitochondrial architecture in the intestine can be categorized manually into fragmented, intermediate and fused network states. (b) Representative pictures showing that loss of raga-1 preserves …
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Figure 4—figure supplement 4—source data 1
Figure 4—figure supplement 4c Network states of intestinal mitochondria in raga-1 mutants.
- https://doi.org/10.7554/eLife.49158.029
Figure 4—figure supplement 5
Neuronal raga-1 expression alters muscle mitochondrial architecture.
Quantification showing that neuronal raga-1 rescue animals also have decreased network count (a) and number of junction points (b) indicating that the mitochondrial architecture in these animals are …
Figure 4—figure supplement 6
Effects of the unc-64 hypomorphic allele on muscle mitochondria morphology.
(a) Representative pictures showing that unc-64(e246) mutants preserve mitochondrial morphology during aging. The mitochondrial backbone (red) is overlaid on binary images. TOMM-20aa1-49::GFP …
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Figure 4—figure supplement 6—source data 1
Figure 4—figure supplement 6c-f Mitochondria network characteristics of muscle mitochondria in unc-64 mutants.
- https://doi.org/10.7554/eLife.49158.030
Figure 5 with 1 supplement
raga-1 deletion requires a fused mitochondrial network to promote longevity.
(a) fzo-1(tm1133) raga-1(ok386) double mutants, which are deficient in mitochondrial fusion, have significantly shortened lifespan compared to raga-1(ok386) single mutants (p<0.0001). However, raga-1…
Figure 5—figure supplement 1
fzo-1(tm1133) suppresses extended lifespan of raga-1(ok386) without the use of FUDR.
The lifespan of fzo-1(tm1133) raga-1(ok386) double mutant is not longer than control animals (p=0.8252). n = 2 independent biological replicates; sample sizes range between 109–153 deaths per …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain (Caenorhabditis elegans) | N2 | Caenorhabditis Genetics Center | WB Cat# N2_(ancestral), RRID:WB-STRAIN:N2_(ancestral) | Laboratory reference strain |
| Strain (C. elegans) | VC222 | Caenorhabditis Genetics Center | WB Cat# VC222, RRID:WB-STRAIN:VC222 | Genotype:raga-1(ok386) II. |
| Strain (C. elegans) | RB754 | Caenorhabditis Genetics Center | WB Cat# RB754, RRID:WB-STRAIN:RB754 | Genotype: aak-2(ok524) X. |
| Strain (C. elegans) | WBM997 | This study | Genotype: aak-2(wbm20) X. | |
| Strain (C. elegans) | RB1206 | Caenorhabditis Genetics Center | WB Cat# RB1206, RRID:WB-STRAIN:RB1206 | Genotype: rsks-1(ok1255) III. |
| Strain (C. elegans) | WBM536 | This study | Genotype: wbmEx238[rab-3p::raga-1 cDNA::SL2::mCherry::unc-54 3'UTR] | |
| Strain (C. elegans) | WBM772 | This study | Genotype: wbmEx333 [rab-3p::rsks-1 cDNA::SL2::mCherry::unc-54 3'UTR] | |
| Strain (C. elegans) | WBM1167 | This study | Genotype: wbmIs79[eft-3p::3XFLAG::raga-1::SL2::wrmScarlet::unc-54 3'UTR, *wbmIs67] | |
| Strain (C. elegans) | WBM1168 | This study | Genotype: wbmIs80[rab-3p::3XFLAG::raga-1::SL2::wrmScarlet::rab-3 3'UTR, *wbmIs68] | |
| Strain (C. elegans) | WBM650 | This study | Genotype: wbmEx271 [ges-1p::raga-1 cDNA::SL2::mCherry::unc-54 3'UTR; rol-6 (su1006)] | |
| Strain (C. elegans) | WBM671 | PMID:29107506 | Genotype:wbmEx289 [myo-3p::tomm20 aa1-49::GFP::unc54 3'UTR] | |
| Strain (C. elegans) | WBM955 | This study | Genotype: wbmEx373 [rab-3p::tomm-20 aa1-49::GFP::unc-54 3'UTR, rol-6] | |
| Strain (C. elegans) | WBM926 | PMID:29107506 | Genotype: wbmEx367[ges-1p::tomm20 aa1-49::GFP::unc-54 3'UTR] | |
| Strain (C. elegans) | CU5991 | Caenorhabditis Genetics Center | WB Cat# CU5991, RRID:WB-STRAIN:CU5991 | Genotype: fzo-1 (tm1133) II. |
| Strain (C. elegans) | CU6372 | Caenorhabditis Genetics Center | WB Cat# CU6372, RRID:WB-STRAIN:CU6372 | Genotype: drp-1(tm1108) IV. |
| Strain (C. elegans) | WBM861 | This study | Genotype: fzo-1(tm1133) II; wbmEx335 [rab-3p:3xFLAG fzo-1 cDNA: unc54 3'UTR, myo-3p:mCherry] | |
| Strain (C. elegans) | WBM612 | PMID:29107506 | Genotype: fzo-1 (tm1133) II; wbmEx258 [pHW11 (myo-3p::3xFLAG::fzo-1 cDNA::unc-54 3'UTR) + pRF4 (rol-6(SU1006))] | |
| Strain (C. elegans) | WBM639 | PMID:29107506 | Genotype: fzo-1(tm1133) II; wbmEx276 [pHW18 (ges-1p::3xFLAG::fzo-1 cDNA::unc54 3’UTR) + pRF4(rol-6(SU1006))] | |
| Antibody | Phospho-AMPKα (Thr172) antibody | Cell Signaling Technology | Cat# 2535, RRID:AB_331250 | |
| Antibody | Beta actin antibody | Abcam | Cat# ab8226, RRID:AB_306371 | |
| Software | MitoMAPR | This study | Source code provided as Source code 1 | |
| Commercial assay or kit | TruSeq Stranded mRNA LT - Set A kit | Illumina | RS-122–2101 |
Additional files
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Source code 1
Code for muscle mitochondria analysis using MitoMAPR.
- https://doi.org/10.7554/eLife.49158.033
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Supplementary file 1
DEGs from pairwise comparisons of RNA-Seq.
- https://doi.org/10.7554/eLife.49158.034
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Supplementary file 2
Genes in each cluster from RNA-seq.
- https://doi.org/10.7554/eLife.49158.035
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Supplementary file 3
GO terms enriched in each cluster.
- https://doi.org/10.7554/eLife.49158.036
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Supplementary file 4
Worm strains.
- https://doi.org/10.7554/eLife.49158.037
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Supplementary file 5
Genotyping strategies.
- https://doi.org/10.7554/eLife.49158.038
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Supplementary file 6
Lifespan replicates.
- https://doi.org/10.7554/eLife.49158.039
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Supplementary file 7
List of MitoMAPR attributes.
- https://doi.org/10.7554/eLife.49158.040
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Transparent reporting form
- https://doi.org/10.7554/eLife.49158.041
